Estrogen receptor alpha regulated gene expression, related assays and therapeutics

ABSTRACT

A plurality of genes modulated by estrogen or other agents, such as hormones or combinations of hormones, in various types of tissue is described. One embodiment of the disclosure relates to a plurality of genes, which demonstrates certain patterns of expression differing qualitatively or quantitatively, with and without exposure to estrogen and/or other hormone compositions. Methods of using these genes in identifying candidate agents that exert at least some of the biological effects of estrogen and/or other hormone, and pharmaceuticals and related therapies also is disclosed. The use of the plurality of genes in methods of monitoring, in gene chips and in kits also is disclosed.

FIELD OF THE INVENTION

The present disclosure relates to a plurality of genes modulated by estrogen or other agents, such as hormones or combinations of hormones, in various types of tissue. In particular, one embodiment of the disclosure relates to a plurality of genes which demonstrates certain patterns of expression differing qualitatively or, quantitatively, with and without exposure to estrogen and/or other hormone compositions. The disclosure further relates to the methods of using these genes in identifying agents that exert at least some of the biological effects of estrogen and/or other agents, and to pharmaceuticals and related therapies. The disclosure further relates to the use of the plurality of genes in methods of monitoring, in gene chips and in kits.

BACKGROUND OF THE INVENTION

Estrogens exert biological effects in numerous organs throughout the body. The role of estrogens in reproductive biology, the prevention of postmenopausal hot flashes, and the prevention of postmenopausal osteoporosis are well established. Many observational studies have suggested estrogens also reduce the risk of development of cardiovascular disease(1), at least in part by estrogens reducing LDL cholesterol levels and elevating HDL cholesterol levels(2,3). More recently, estrogens have been suggested to inhibit the development of colon cancer(4), inhibit the development of Alzheimer's disease(5), and inhibit development of cataracts (6). The multitude of estrogen responses matches the widespread distribution of estrogen receptors (ER) throughout numerous organs, with ERα expression highest in uterus, pituitary, kidney and adrenal gland and ERβ expression highest in ovary, uterus, bladder and lung(7). While various estrogens have been profiled for biological activity, little is known regarding the patterns of gene expression which are responsible for these diverse activities.

Thus, a need exists for the systemic analysis of the regulation by estrogen and/or other hormonal compositions of gene expression in various tissues and the identification of the plurality of differentially expressed genes. The identification of candidate agents that at least partially exert the same differential expression and development of pharmaceuticals and new treatment methods based on such agents is highly desirable. There also exists a need for methods of monitoring conditions and for diagnostic products, including gene chips and kits, which may be used in the above-described analyses.

The embodiments provided herein relate generally to a plurality of genes, particularly a plurality of genes that are modulated by estrogen and/or other hormonal compositions in various organs, such as the uterus, kidney and pituitary gland. Such differentially expressed genes are useful in screening assays to examine the effects of a candidate agent on the expression of genes that are responsive to estrogen. A candidate agent that induces, in a given tissue, a gene expression profile that exhibits one or more similarities to the gene expression profile of estrogen and/or other hormonal compositions, can be identified for possible use in pharmaceuticals. The invention also relates to the identification of estrogen responsive genes that are known to be associated with the inhibition of certain conditions, such as shock, post-menopausal calcium deficiencies, cardiovascular diseases, and conditions where there is decreased renal blood flow, such as those caused by diuretics or congestive heart failure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a pattern analysis generated by a GeneChip microarray analysis. Specifically, WT or ERβKO ovariectomized mice were treated daily with vehicle or 20 μg/kg/day E2 for six weeks. Two hours following the final dose, the mice were euthanized and 13 tissues removed for RNA preparation. Two independent studies were performed, with total RNA pooled from two groups of three animals for each condition. Gene expression was quantified by GeneChip microarrays using murine U74 sub A arrays. Data were analyzed for patterns indicating either ERα or ERβ dependent regulation as shown. For ERα regulation, the defined search patterns (induction or repression) were for regulation by E2 in both wild type and ERβKO mice, in both sets of mice in both studies. For ERβ regulation, the defined search patterns were for regulation by E2 only in the wild type mice, with no change in basal expression in the ERβKO mice compared to the wild type mice. The number of genes in each tissue that matched the theoretical induction (↑) or repression (↓) patterns for ERα or ERβ are indicated.

FIGS. 2A-2C show a series of bar graphs showing gene expression levels of known genes regulated in the kidney in an ERα pattern. The expression levels (parts-per-million) are shown for the indicated genes in WT mice treated with vehicle (light blue bars), WT mice treated with E2 (dark blue bars), ERβKO mice treated with vehicle (light green bars), and ERβKO mice treated with E2 (dark green bars) using U74v2 subA, BS and C microarrays. Expression was measured in two independent sets of animals, with two groups of animals for each treatment in each study. A gene name abbreviation is shown above each graph, with the corresponding Unigene designation shown below. The genes are graphed in approximate order of regulation from largest induction (CYP7B1) to largest repression (BHMT).

FIG. 3 shows histological sections of the kidney in in situ hybridization studies using antisense probes for CYP7B1, TF, STAT5A or GADD45G in ovariectomized mice treated with vehicle or 20 μg/kg/day E2 for six weeks. No signal was detected with the corresponding sense probes.

FIG. 4 shows histological sections of the kidney in in situ hybridization using antisense probes for STAT5A or GAD45G in ovariectomized rats treated with vehicle or 20 μg/kg/day E2 for six weeks. No signal was detected with the corresponding sense probes.

FIG. 5A-5B show a series of graphs showing expression levels for various genes. Ovariectomized WT mice were treated with vehicle or various doses of E2 for six weeks. (A) Kidney gene expression values (mean±SEM) were determined by real-time PCR for each individual animal and normalized for GAPDH expression. The mean expression level in vehicle-treated mice was defined as 1 for each gene. (B) Uterine wet weights (mg) and gene expression values (mean t SEM).

FIGS. 6A-6D show a series of bar graphs depicting relative expression levels for various genes. Ovariectomized WT mice were treated with vehicle, 20 μg/kg/day E2, 5 mg/kg/day W-0292, W-0070 or propylpyrazole triol (PPT) for six weeks. Kidney gene expression values were determined by real-time PCR for each individual animal and normalized for GAPDH expression. The mean expression level in vehicle-treated mice was defined as 1 for each gene. *p<0.01 for comparison to vehicle treated animals.

FIGS. 7A-7D show bar graphs depicting expression levels of intact and ΔAF1-ERα mRNA determined in uterus and kidney by using a real-time PCR assay specific for exon 3 of the mouse ERα or ERβ genes. Each graph utilizes a different scale. Expression levels were normalized for total RNA level to avoid GAPDH expression differences between kidney and uterus.

FIGS. 8A-BC show a series of relative expression levels for various genes in different types of mice. This figure also presents a model for AF1 or AF2 activation for each gene. Ovariectomized WT mice, ERαERβKO mice (expressing only ΔAF1-ERα) or ERαKO mice (expressing ΔAF1-ERα along with ERβ) were treated for 6 weeks with vehicle, 10 μg/kg/day E2, 10 μg/kg/day E2+5 mg/kg/day ICI182780, or 5 mg/kg/day tamoxifen. Kidney gene expression values were determined by real-time PCR for each individual animal and normalized for GAPDH expression. The mean expression level in vehicle-treated WT mice was defined as 1 for each gene. *p<0.01 for comparison to vehicle treated animals. A model for the requirement of AF1 or AF2 for activation of each gene is shown below each graph. The change in ER shape with tamoxifen Cr) bound denotes the alternate helix 12 conformation induced by tamoxifen compared to E2. CA denotes coactivators.

SUMMARY OF EMBODIMENTS

One embodiment of the disclosure relates to a plurality of genes, each of whom is differentially expressed in tissue cells exposed to estrogen and/or other hormones or combination of hormones and tissue cells without said exposure, which plurality comprises a first group and a second group, wherein each gene in said first group is differentially expressed at a higher level in said tissue cells exposed to estrogen and/or a hormone or combinations of hormones than in said tissue cells without said exposure, wherein each gene in said second group is differentially expressed at a lower level in said tissue cells exposed to estrogen and/or a hormone or combinations of hormones than in said tissue cells without said exposure. Confirmation of such expression is confirmed by real-time PCR. Such cells preferably are from the kidney, pituitary or uterus. Exposure to estrogen and/or the other hormones is in vivo or in vitro. The higher level and lower levels are assessed using a predetermined statistical significance standard based on measurements of expression levels. The measurements can obtained using nucleotide arrays or nucleotide filters.

Another embodiment relates to a method for identifying an agent having the biological effect of estrogen and/or other hormones or combination of hormones, on gene expression in a given tissue, wherein said desired effect represents a first plurality of genes differentially expressed at various levels, which method comprises:

-   exposing, in vivo or in vitro, tissue cells to said agent; -   measuring expression levels of a multiplicity of genes in said     tissue cells exposed to said agent and tissue cells without said     exposure, said multiplicity being greater than said first plurality; -   determining, using a predetermined statistical significance     standard, genes which are differentially expressed in said tissue     cells exposed to said agent and said tissue cells without said     exposure, said genes constitute a second plurality; and -   comparing the expression levels of genes in said second plurality     with the expression levels of genes in said first plurality,     wherein said agent is identified as having said desired effort if     said first and second pluralities are the same and said expression     levels in said first and second pluralities are substantially the     same. The tissue preferably is kidney, uterus or pituitary tissue.     Expression levels are confirmed by real-time PCR.

Another embodiment is directed to an agent identified by the above method.

Another embodiment is a pharmaceutical composition comprising this agent and a pharmaceutically acceptable excipient.

Another embodiment relates to a method for identifying an agent capable of maintaining vascular volume in septic shock, which method comprises:

-   exposing, in vivo or in vitro, kidney cells to the agent; -   measuring expression levels of NTT73 and ABCC3 in said kidney cells     exposed to the agent and kidney cells without the exposure; -   comparing the expression levels of NTT73 and ABCC3 with the     expression levels of genes in the plurality if genes described above     with regard to the kidney, wherein the induced genes are NTT73 and     ABCC3,     wherein said agent is identified as capable of maintaining vascular     volume in septic shock if said expression levels of NTT73 and ABCC3     are substantially the same as said expression levels of genes in     such plurality.

Another embodiment relates to a method of identifying an agent capable of enhancing calcium uptake in post-menopausal women, which method comprises:

-   exposing, in vivo or in vitro, kidney cells to said agent; -   measuring expression levels of CYP7B1 in said kidney cells exposed     to said agent and kidney cells without said exposure; -   comparing the expression levels of CYP7B1 with the expression levels     of genes in the plurality of genes in the kidney is induced CYP7B1,     wherein said agent is identified as capable of enhancing calcium     uptake in post-menopausal women if said expression levels of CYP7B1     are substantially the same as said expression levels of genes in     such plurality.

Another embodiment relates to a method for identifying an agent for treating cardiovascular disorders, which method comprises:

-   exposing, in vivo or in vitro, kidney cells to said agent; -   measuring expression levels of BHMT and SAHH in said kidney cells     exposed to said agent and kidney cells without said exposure; -   comparing the expression levels of BHMT and SAHH with the expression     levels of genes in the plurality of genes, wherein in the kidney     BHMT and SAHH are repressed,     wherein said agent is identified for treating cardiovascular     disorders if said expression levels of BHMT and SAHH are     substantially the same as said expression levels of genes in the     such plurality.

Another embodiment relates to agents identified by any of the above methods and pharmaceutical agents comprising such agents and a pharmaceutically acceptable excipient.

Another embodiment relates to a solid substrate comprising any of the above described plurality of genes.

Another embodiment relates to a kit comprising any of the above plurality of genes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The embodiments herein provide a plurality of genes modulated by estrogen and/or other hormonal compositions of interest in various types of tissue and the use of such a plurality of differentially expressed genes in screening for agents that exert at least some of the biological effects of estrogen and other hormonal compositions of interest. Such identified agents can be used in pharmaceuticals and in related new therapeutic methods. The plurality of genes can be used in methods of monitoring.

Definitions:

In general, “a gene” is a region on the genome that is capable of being transcribed to an RNA that either has a regulatory function, a catalytic function and/or encodes a protein. A gene typically has introns and exons, which may organize to produce different RNA splice variants that encode alternative versions of a mature protein. “Gene” contemplates fragments of genes that may or may not represent a functional domain.

A “plurality of genes” as used herein refers to a group of identified or isolated genes whose levels of expression vary in different tissues, cells or under different conditions or biological states. The different conditions may be caused by exposure to certain agent(s)—whether exogenous or endogenous—which include hormones, receptor ligands, chemical compounds, etc. The expression of a plurality of genes demonstrates certain patterns. That is, each gene in the plurality is expressed differently in different conditions or with or without exposure to a certain endogenous or exogenous agents. The extent or level of differential expression of each gene may vary in the plurality and may be determined qualitatively and/or quantitatively according to this invention. A gene expression profile, as used herein, refers to a plurality of genes that are differentially expressed at different levels, which constitutes a “pattern” or a “profile.” As used herein, the term “expression profile,” “profile,” “expression pattern,” “pattern,” “gene expression profile,” and “gene expression pattern” are used interchangeably.

An “agent that exerts at least some of the biological effects of estrogen,” as used herein refers to any factor, agent, compound whether endogenous or exogenous in origin, which is capable of binding and interacting with estrogen receptors and thereby eliciting certain biological effects of extrogen. The skilled artisan would know that, for instance, one of the biological effects of estrogen is to promote the development of the female reproductive system. Other biological effects of estrogen are well documented and discussed, infra.

Gene expression profiles may be measured, according to this invention, by using nucleotide or microarrays. These arrays allow tens of thousands of genes to be surveyed at the same time.

“Hormones or combinations of hormones” include for instance, combinations of estrogens or other hormones that are known to exert biological effects of estrogen.

As used herein, the term “microarray” refers to nucleotide arrays that can be used to detect biomolecules, for instance to measure gene expression. “Array,” “slide,” and “chip” are used interchangeably in this disclosure. Various kinds of arrays are made in research and manufacturing facilities worldwide, some of which are available commercially. There are, for example, two main kinds of nucleotide arrays that differ in the manner in which the nucleic acid materials are placed onto the array substrate: spotted arrays and in situ synthesized arrays. One of the most widely used oligonucleotide arrays is GeneChip™ made by Affymetrix, Inc. The oligonucleotide probes that are 20- or 25-base long are synthesized in silico on the array substrate. These arrays tend to achieve high densities (e.g., more than 40,000 genes per cm²). The spotted arrays, on the other hand, tend to have lower densities, but the probes, typically partial cDNA molecules, usually are much longer than 20- or 25-mers. A representative type of spotted cDNA array is LifeArray made by Incyte Genomics. Pre-synthesized and amplified cDNA sequences are attached to the substrate of these kinds of arrays.

In one embodiment, the nucleotide is an array (i.e., a matrix) in which each position represents a discrete binding site for a product encoded by a gene (e.g., a protein or RNA), and in which binding sites are present for products of most or almost all of the genes in the organism's genome. In one embodiment, the “binding site” (hereinafter, “site”) is a nucleic acid or nucleic acid analogue to which a particular cognate cDNA can specifically hybridize. The nucleic acid or analogue of the binding site can be, e.g., a synthetic oligomer, a full-length cDNA, a less-than full length cDNA, or a gene fragment.

Although the microarray may contain binding sites for products of all or almost all genes in the target organism's genome, such comprehensiveness is not necessarily required. Usually the microarray will have binding sites corresponding to at least about 50% of the genes in the genome, often at least about 75%, more often at least about 85%, even more often more than about 90%, and most often at least about 99%. Preferably, the microarray has binding sites for genes relevant to the action of the gene expression modulating agent of interest or in a biological pathway of interest.

The nucleic acid or analogue are attached to a “solid support,” which may be made from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, or other materials. A preferred method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al., 1995, Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science 270:467-470. This method is especially useful for preparing microarrays of cDNA. See also DeRisi et al., 1996, Use of a cDNA microarray to analyze gene expression patterns in human cancer, Nature Genetics 14:457-460; Shalon et al., 1996, A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization, Genome Res. 6:639-645; and Schena et al., 1995, Parallel human genome analysis; microarray-based expression of 1000 genes, Proc. Natl. Acad. Sci. USA 93:10539-11286.

In a preferred embodiment, the microarray is a high-density oligonucleotide array, as described above. In a particularly preferred embodiment, the nucleotide arrays are the MG_U74 and MG_U74v2 arrays from Affymetrix.

“Polymerase Chain Reaction” or “PCR” is an amplification-based assay used to measure the copy number of the gene. In such assays, the corresponding nucleic acid sequences act as a template in an amplification reaction. In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the copy number of the gene, corresponding to the specific probe used, according to the principle discussed above. Methods of “real-time quantitative PCR” using Taqman probes are well known in the art. Detailed protocols for real-time quantitative PCR are provided, for example, for RNA in: Gibson et al., 1996, A novel method for real time quantitative RT-PCR. Genome Res. 10:995-1001; and for DNA in: Heid et al., 1996, Real time quantitative PCR. Genome Res. 10:986-994.

A TaqMan-based assay can also be used to quantify polynucleotides. TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5′ fluorescent dye and a 3′ quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3′ end. When the PCR product is amplified in subsequent cycles, the 5′ nuclease activity of the polymerase, for example, AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5′ fluorescent dye and the 3′ quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, for example, http://www2.perkin-elmer.com).

Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see, Wu and Wallace, 1989, Genomics 4: 560; Landegren et al., 1988 Science 241: 1077; and Barringer et al., 1990, Gene 89: 117), transcription amplification (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli et al., 1990, Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.

The “level of mRNA” in a biological sample refers to the amount of mRNA transcribed from a given gene that is present in a cell or a biological sample. One aspect of the biological state of a biological sample (e.g. a cell or cell culture) usefully measured in the present invention is its transcriptional state. The transcriptional state of a biological sample includes the identities and abundances of the constituent RNA species, especially mRNAs, in the cell under a given set of conditions. Preferably, a substantial fraction of all constituent RNA species in the biological sample are measured, but at least a sufficient fraction is measured to characterize the action of an agent or gene modulator of interest. The level of mRNA may be quantified by methods described herein or may be simply detected, by visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.

A “biological sample,” as used herein refers to any sample taken from a biological subject, in vivo or in situ. A biological sample may be a sample of biological tissue, or cells or a biological fluid. Biological samples may be taken, according to this invention, from any kind of biological species, any types of tissues, and any types of cells, among other things. Cell samples may be isolated cells, primary cell cultures, or cultured cell lines according to this invention. Biological samples may be combined or pooled as needed in various embodimets. Preferred tissues are from the uterus, kidney, pituitary glands, breast, brain and adipose tissue.

“Modulation of gene expression,” as this term is used herein, refers to the induction or inhibition of expression of a gene. Such modulation may be assessed or measured by assays. Typically, modulation of gene expression may be caused by endogenous or exogenous factors or agents. The effect of a given compound can be measured by any means known to those skilled in the art. For example, expression levels may be measured by PCR, Northern blotting, Primer Extension, Differential Display techniques, etc.

“Induction of expression” as used herein refers to any observable or measurable increase in the levels of expression of a particular gene, either qualitatively or quantitatively. The measurement of levels of expression may be carried out according to this invention using any techniques that are capable of measuring RNA transcripts in a biological sample. Examples of these techniques include, as discussed above, PCR, TaqMan, Primer Extension, Differential display and nucleotide arrays, among other things.

“Repression of expression.” “Repression” or “inhibition” of expression, are used interchangeably according to this disclosure. It refers to any observable or measurable decrease in the levels of expression of a particular gene, either qualitatively or quantitatively. The measurement of levels of expression may be carried out using any techniques that are capable of measuring RNA transcripts in a biological sample. The examples of these techniques include, as discussed above, PCR, TaqMan, Primer Extension, Differential Display, and nucleotide arrays, among other things.”

A “gene chip” or “DNA chip” is described, for instance, in U.S. Pat. Nos. 5,412,087, 5,445,934 and 5,744,305 and is useful for screening gene expression at the mRNA level. Gene chips are commercially available.

A “kit” is one or more of containers or packages, containing at least one “plurality of genes,” as described above, on a solid support. Such kit also may contain various reagents or solutions, as well as instructions for use and labels.

A “detectable label” or a “detectable moiety” is a composition that when linked with a nucleic acid or a protein molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenenin or haptens. A “labeled nucleic acid or oligonucleotide probe” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently through ionic, vander Waals, electrostatic, hydrophobic interactions, or hydrogen bonds, to a label such that the presence of the nucleic acid or probe may be detected by detecting the presence of the label bound to the nucleic acid or probe.

A “nucleic acid probe” is a nucleic acid capable of binding to a target nucleic acid or complementary sequence through one or more types of chemical bond, usually through complementary base pairing usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. It will be understood by one of skill in the art that probes may bind target sequences that lack complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled with isotopes, for example, chromophores, luminphores, chromogens, or indirectly labeled with biotin to which a strepavidin complex may later bind. By assaying the presence or absence of the probe, one can detect the presence or absence of a target gene of interest.

“In situ hybridization” is a methodology for determining the presence of or the copy number of a gene in a sample, for example, fluorescence in situ hybridization (FISH) (see Angerer, 1987 Meth. Enzymol 152: 649). Generally, in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target nucleic acid, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization, and (5) detection of the hybridized nucleic acid fragments. The probes used in such applications are typically labeled, for example, with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions.

Hybridization protocols suitable for use with the methods of the invention are described, for example, in Albertson (1984) EMBO J. 3:1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85:9138-9142; EPO Pub. No. 430:402; Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994); etc.

“A predetermined statistical significance standard based on measurements of expression levels” is a confidence score based upon the assessment of four factors. Specifically, a score is assigned to each gene that reflects the confidence of the change. The score is based on four criteria: Fold Change, p-value (T-test), Present Calls, Frequency Value. For example, see the Table below: TABLE I Confidence criteria Score Fold Change >2.0 5 >1.5 0 <1.5 −3 pValue  <0.05 3 0.05 to 0.1  2 0.1 to 0.2 0 0.2 to 0.3 −1 0.3 to 0.5 −3 Present Calls 2-4 3  1  1  0  0 Second Largest Frequency >20   3  15 to 191 10-14 −1 <10   −3 Outliers (Max Freq/2^(nd) largest) >2.5 −3

“Data” refers to information obtained that relates to the expression of genes in response to exposure to estrogen or an agent of unknown biological effect. The plurality of genes identified by the disclosed methods are examples of such information. The information is stored in electronic or paper formats. Electronic format can be selected from the group consisting of electronic mail, disk, compact disk (CD) digital versatile disk (DVD), memory card, memory chip, ROM or RAM, magnetic optical disk, tape, video, video clip, microfilm, internet, shared network, shared server and the like; wherein data is displayed, transmitted or analyzed via electronic transmission, video display, telecommunication, or by using any of the above stored formats; wherein data is compared and compiled at the site of sampling specimens or at a location where the data is transported following a process as described above.

“Genes modulated by estrogen.” Genes regulated by estrogen and/or hormonal compositions and identified according to the disclosed methods, are listed in Tables II, III and IV. Relevant Unigene codes or Genbank accession numbers are provided.

Identification of Genes Modulated by Estrogen

A. Biological Sample and Assay

One embodiment disclosed herein relates to a plurality of genes, each of which is differentially expressed in kidney cells exposed to estrogen or a candidate agent and kidney cells without exposure to estrogen or a candidate agent, which plurality comprises a first group and a second group, wherein each gene in said first group is differentially expressed at a higher level in said kidney cells exposed to estrogen or a candidate agent than in said kidney cells without said exposure, wherein each gene in the second group is differentially expressed at a lower level in said kidney cells exposed to estrogen or candidate agent than in said kidney cells without said exposure.

A biological sample of kidney cells are obtained according to methods well known to the skilled artisan. One group of kidney cells are exposed to estrogen. Such estrogen may be 17β estradiol. The kidney cells may be from one or more animals of the same species or from a culture of kidney cells or kidney tissue. Preferably, such cells are from a mammal, most preferably a mouse, rat or human. Such animal must produce little or no estrogen. For instance, an aromatase knockout animal cannot produce estrogen. Because the major source of circulating estrogen is the ovary, ovariectomy dramatically decreases circulating estrogen levels. Thus, in one embodiment, ovariectomized animals are used. By “exposure” is meant a type and quantity of either in vivo or in vitro administration that is applicable to the source of the kidney cells and known and acceptable to those of skill of the art. The total RNA from such cells is prepared by methods known to the skilled artisan, e.g., by Trizol (Invitrogen) followed by subsequent repurification, e.g. via Rneasy columns (Qiagen). The total RNA is used to generate a labeled target according to methods and using detectable labels well know in the art, as described above in detail. For instance, the RNA may be labeled with biotin to form a cRNA target for use in an assay. See a complete description of preferred methods in the Affymetrix GeneChip® technical manual (Pages 700217 through 700223), which is herein incorporated by reference.

The assay, according to the invention, may be any assay suitable to detect gene expression. For instance, mRNA, cDNA or protein expression may be detected. Many different types of assays are known, examples of which are set forth above, including analyses by nucleotide arrays and nucleotide filters. The hybridization conditions (temperature, time, and concentrations) are adjusted according to procedures also well known in the art, as described above. In a preferred embodiment, the assay of the invention involves the use of a high density oligonucleotide array. For instance, in a preferred embodiment, cRNA labeled with biotin is hybridized to a murine MG_U74Av2 probe array (Affymetrix, Santa Clara, Calif.) for 16 hours at 45 degrees. Eleven biotin-labeled cRNAs at defined concentration are spiked into each hybridization and used to convert average difference values to frequencies expressed as parts per million.

Other solid supports and microarrays are known and commercially available to the skilled artisan, as described above.

B. Measurements and Statistical Analysis

The assay of the invention is used to identify genes modulated by estrogen. Such modulation may be induction of expression (a plurality of genes belonging to a “first group”) or repression of such expression (a plurality of genes belonging to a “second group”). Gene expression induction is indicated by a higher level of expression, whereas repression is indicated by a lower level of expression, as assessed using a predetermined statistical significance standard based on measurements of expression levels.

Thus, the genes expressed or repressed in kidney cells with estrogen exposure are compared to the genes expressed or repressed in kidney cells that were not exposed to estrogen. Pairwise comparisons are made between each of the treatments. A pairwise comparison is the expression data for a given gene under a given treatment condition compared to the expression data for this gene under a second treatment condition. The fold change ratio is then calculated, the p-value based on Student's t-test, the number of present calls, and the expression level for each comparison. A confidence score “CS” is defined as CS(x)=FC(x)+PV(x)+PC(x)+EL(x) where FC, PV, PC and EL are scores assigned to the fold change, p-value, number of present calls, and the expression level, respectively. FC(x) is assigned 5 if the fold change ratio was greater then 1.95 and is assigned 0 if the ratio is between 1.95 and 1.5. PV(x) is assigned 3 if the p-value is less then 0.05 and is assigned 2 if the p-value was between 0.05 and 0.1. PC(x) is assigned 3 if at least 50% of the samples are called P by the Affymetrix algorithm and assigned 1 if only 25% of the samples are called P. EL(x) is assigned 3 if at least two samples have frequency value of 20 or greater and assigned 1 if two samples only have a frequency greater then 15. Penalty points are assigned if the fold change is less then 1.5, the p-value is greater then 0.2 or the frequency values were below 15 ppm. CS(x) ranged for −14 to 14 with qualifiers having a score of 14 considered the most significant changes. Genes with 11 or more points in any one pairwise comparison is considered to be significant. Real-time PCR and histology analyses are then performed to confirm the identity of the genes, essentially as described previously (9,10), which are herein incorporated by reference. The above described analysis can be used to identify candidate agents that are “estrogen-like” in that they have a differential expression profile which is in the most preferred embodiment substantially the same as estrogen's. For instance, in one embodiment, the expression levels for the genes upon exposure to the respective compounds is at least within 50% of each other.

C. Biological Samples from Other Organs

The above described methods, assay and analysis can be applied to biological samples from any tissue, including the uterus, pituitary gland, liver, brain, colon, breast, adipose tissue, etc. In preferred embodiments, the biological samples are from the kidney, pituitary gland and the uterus.

D. The Plurality of Genes

Pursuant to the above described methods, the genes listed in Table II were identified as being differentially expressed upon exposure to estrogen. Genes in which expression is induced by estrogen are considered to be genes of the “first group,” whereas genes that are repressed by estrogen are considered to be in the “second group”.

Specifically, the estrogen modulated genes in the kidney are Tissue Factor, CYP7B1, BCAT1, STAT5A, GADD45G, BHMT, SAHH, NTT73, ABCC3. Of these genes, estrogen induced expression in all but BHMT and SAHH, where it repressed expression.

Thus, one disclosed embodiment is a plurality of genes, wherein in the first group, where gene expression in kidney cells is induced by estrogen exposure, the plurality of genes comprise NTT73 and ABCC3. Another disclosed embodiment is a plurality of genes wherein the “first group” comprises CYP7B1 in kidney cells. In another embodiment, the plurality of genes of the “second group,” where gene expression in kidney cells is repressed by estrogen exposure, comprises at least BHMT and SAHH.

Another disclosed embodiment is directed to a plurality of genes in kidney cells, wherein the first group comprises Tissue Factor, CYP7B1, BCAT1, STAT5A, and GADD45G, and wherein said second group comprises BHMT.

Another disclosed embodiment is directed to a plurality of the genes wherein the first group comprises CYP7B1, TF, SCYA28, Iga, Vk28, PHD 2, ELF 3, TIM1, STAT5A, COR1, BCAT1, ABCC3, TIM2, NAT6, RGS3, GNBP3, BCL7A, 17βDHH, FYVE ZFP, NTT73, AGPS, TRIM2, HBACH, CIS2, CYP27B1, and STAT5B, wherein said second group comprises SAHH, ADH1A7, RARRES2, and BHMT. Another disclosed embodiment relates to a plurality of genes, wherein the first group comprises COR1 and GNBP3.

The estrogen modulated genes in the pituitary gland are STAT5B, GADD45G, Kallikrein-9, and FSHb, the expression of which is induced by estrogen for all but FSHb, which is repressed.

Thus one embodiment relates to a plurality of genes in the pituitary gland, wherein the first group comprises STAT5B and GADD45G.

Another embodiment relates to a plurality of genes, wherein the first group comprises STAT5B, GADD45G1 and Kallikreins genes in the pituitary.

Yet another embodiment relates to a plurality of genes, wherein the second group of genes in the pituitary gland comprise FSHb.

Pursuant to the above methods, the inventors discovered that the estrogen modulated genes in the uterus comprise SFRP4, Deiodinase (type II), Procollagen (type I, Alpha I), vimentin and IGFBP4, Scavenger receptor, AI121305, ALOX15, BCAT1, SiAMOX, C3, FOS, MAP2k1, CEBPb, EGR1 and CYP1A1. All of these genes are induced by estrogen in the uterus except for Scavenger receptor and CYP1A1, which are repressed.

Thus, one embodiment is directed to a plurality of genes in the uterus, wherein the first group comprises SFRP4, Deiodinase (type II), Procollagen (type I, Alpha I), vimentin and IGFBP4.

Another embodiment of the invention is directed to the plurality of genes, wherein the first group in the uterus comprises AI121305, ALOX15, BCAT1, SiAMOX, C3, FOX, MAP2k1, CEBPb and EGR1.

Another embodiment is directed to a plurality of genes wherein the first group in the uterus comprises SFRP4, Deiodinase (type II), Procollagen (type I, Alpha I), vimentin and IGFBP4, Scavenger receptor, AI121305, ALOX15, BCAT1, SiAMOX, C3, FOS, MAP2k1, CEBPb and EGR1.

In another embodiment, the plurality of genes in the second group in the uterus comprises CYP1A1.

In yet another embodiment, the plurality of genes in the second group in the uterus comprises Scavenger receptor.

Methods of Identifying Agents

Based upon the above described methods for determining differential expression of genes in various organs, another aspect of the invention relates to the identification of candidate agents that have the same or substantially the same biological effect of a known agent, such as estrogen or another hormonal combination of known biological effect. An “agent” could be any compound of unknown biological effect on genes in a given body site. Specifically, the invention relates to a method for identifying an agent having a desired effect on gene expression in an organ, wherein said desired effect represents a first plurality of genes differentially expressed at various levels, which method comprises:

-   -   exposing, in vivo or in vitro, organ cells to the agent;     -   measuring expression levels of a multiplicity of genes in the         organ cells exposed to the agent and organ cells without the         exposure, the multiplicity being greater than said first         plurality;     -   determining, using a predetermined statistical significance         standard, genes which are differentially expressed in the organ         cells exposed to the agent and the organ cells without the         exposure, the genes constitute a second plurality; and     -   comparing the expression levels of genes in the second plurality         with the expression levels of genes in said first plurality,         wherein the agent is identified as having said desired effort if         said first and second pluralities are the same and said         expression levels in said first and second pluralities are         substantially the same. The “organ cells” may be from any type         of biological sample, as described above. In a preferred         embodiment, such cells are from the kidney, pituitary gland or         uterus. The “first plurality of genes” and “second plurality” of         genes can be identified through a nucleotide array or filter, as         described above. The comparing is performed using a suitable         statistical technique with the assistance of known and         commercially available programs, also as described above.

Another embodiment relates to an agent identified by the above method.

Yet another embodiment relates to a gene chip comprising any one or more of the above plurality of genes.

Pharmaceuticals and Methods of Treating

The identification of agents that induce or repress the expression of a gene associated with a given disorder or condition can lead to the development of pharmaceuticals that can be administered to a patient at therapeutically effective doses to prevent, treat, or control such disorder or condition.

Some conditions associated with estrogen regulation of gene expression in the kidney are known. For instance, in women, high estrogen levels preceding ovulation, during pregnancy, and resulting from estrogen administration commonly results in body water retention (23,24). Increased renal sodium reabsorption is a major mechanistic component for the elevated fluid retention (25). In rats, estrogen has been shown to increase thiazide-sensitive NaCl cotransporter expression levels(26), providing one possible molecular basis for estrogen effects on sodium retention.

Pursuant to the methods of the invention as disclosed above and as exemplified in greater detail in the Examples below, two additional estrogen regulated genes that influence sodium retention were identified. First, estrogen (E2) treatment increased mRNA levels for NTT73 (27), which is a sodium and chloride dependent transporter, known to regulate sodium retention. Second, E2 treatment also induced mRNA levels for ABCC3, a member of a family of genes which are known to modulate epithelial sodium channel activity (28). The physiological role of E2 regulation of these genes may lie in the large volume expansion required during pregnancy. The ED50 value for E2 activation of gene expression in the kidney was about 10-fold higher than that required for uterine weight increases (FIG. 5), perhaps a mechanism to ensure that normally estrogen actions only occur in the kidney when very high levels of estrogen are present, as during pregnancy.

Premenopausal women survive septic shock better than comparably aged males while postmenopausal women have a diminished survival advantage. Since volume loss is a major cause of morbidity in shock, it is expected that enhanced sodium and water retention due to elevated expression of NTT73(27) by E2 plays a role in this protective process. Thus, one disclosed embodiment relates to a method for identifying an agent capable of maintaining vascular volume in septic shock comprising exposing, in vivo or in vitro, kidney cells to an agent, measuring expression levels of NTT73 and ABCC3 in kidney cells exposed the agent and in kidney cells not exposed to the agent; comparing the expression levels of the NTT73 and ABCC3 with the expression levels of the genes kidney cells exposed to estrogen. The candidate agent identified by this process can be used in pharmaceuticals for purposes of maintaining vascular volume in the treatment of septic shock.

Another estrogen modulated gene in the kidney with biological significance is CYP27B1, the enzyme responsible for the rate limiting conversion of inactive 25-hydroxy vitamin D3 into active 1,25-dihidroxy vitamin D (29). This process is known to occur in the proximal tubules of the kidney and has been shown to be stimulated by estrogen treatment of birds(30). Urinary calcium excretion is increased in postmenopausal women, while estrogen treatment reduces urine calcium levels (31, 32). The presence of vitamin D receptors within the proximal convoluted tubule and collecting duct tubules of the kidney suggests that E2 induction of CYP27B1 is the basis of this beneficial effect.

Thus, the invention relates to a method of identifying agents that are capable of enhancing calcium uptake in postmenopausal women comprising exposing, in vivo or in vitro, kidney cells to an agent, measuring expression levels of CYP7B1 in kidney cells exposed the agent and in kidney cells not exposed to the agent; comparing the expression levels of the CYP7B1 with the expression levels of the genes in kidney cells exposed to estrogen. The agent identified by this process can be used in pharmaceuticals for purposes of enhancing calcium uptake in postmenopausal women.

It is known that estrogen treatment reduces expression of betaine:homocysteine methyltransferase (BHMT) and S-adenosylhomocysteine hydrolase (SAHH), two enzymes involved in the methionine/homocysteine cycle (34). Elevated plasma homocysteine levels are now recognized as an important risk factor for the development of cardiovascular disease (35), and estrogen treatments reduced plasma homocysteine levels in postmenopausal women. Thus, the regulation of BHMT and SAHH provides a mechanistic link for this effect.

Thus, one embodiment disclosed herein relates to a method of identifying candidate agents for treating cardiovascular disorders comprising measuring expression of BHMT and SAHH in kidney cells exposed to an agent and in kidney cells with such exposure, comparing the expression levels of BHMT and SAHH with the expression levels of the genes in kidney cells exposed to estrogen. The agent identified by this process can be used in pharmaceuticals for purposes of treating cardiovascular disorders.

Finally, E2 treatment induced expression of COR1 (chemokine orphan receptor 1, RDC1) an orphan G-protein coupled receptor (37), along with the guanylate nucleotide binding protein 3 (GNBP3) and the regulator of G-protein signaling 3 (RGS3), suggesting these proteins may form a functional unit. RDC1 is a receptor for the potent vasodilatory peptide adrenomedullin and calcitonin gene-related peptide, CGRP (38). Administration of CGRP to ovariectomized rats does not produce a decrease in kidney vascular resistance; however, in ovariectomized rats treated with E2 or in pregnant rats, injection of CGRP significantly decreases kidney vascular resistance (39). The observed increased expression of RDC1 in kidney provides a mechanism for the E2 induction of sensitivity to CGRP in the kidney, resulting in the large increase in renal flow seen during pregnancy (40).

Thus, one embodiment disclosed herein relates to a method of identifying candidate agents for treating conditions associated with reduced renal flow, such as caused by diuretics or congestive heart failure. Toxicity and therapeutic efficacy of such agents identified by the above methods can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed by the ratio, LD50/ED50. compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to normal cells and thereby reduce side effects.

The data obtained from the cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds likes preferably within a range of circulating concentrations that include ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration.

The pharmaceuticals of the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients and the biologically active agent. The agents and its physiologically acceptable salts and solvates can be formulated and administered orally, introraly, rectally, parenteraly, epicutaneously, topically, transdermally, subcutaneously, intramuscularly, intranasally, sublingually, intradurally, introcularly, intravenously, intraperioneally, or by inhalation.

With regard to oral administration, the pharmaceutical compositions can take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipient, such as binding agents etc. Tablets may be coated according to methods well known in the art. Liquid preparations can be in the form of solutions, suspensions and syrups or can be initially in dry form for constitution with water or other suitable vehicle. Other additives may include suspending agents, such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats, emulsifying agents or non-aqueous solutions. Preparations for oral administration may also be formulated for a time or controlled release of the active ingredient using techniques well know in the art of the invention.

Other formulations of the pharmaceuticals of the invention may be depot preparations for administration via implantation.

The pharmaceutical compositions of the present invention may be presented in a pack or dispenser device that contains one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, for example a blister pack. The pack or dispenser would contain instructions for administration.

Methods of Monitoring

The identification of the plurality of genes described above provides a powerful tool for assessing the progression of a state, condition or treatment. Specifically, a plurality of genes can be identified in a patient prior to an event, such as menopause, surgery, the onset of a therapeutic regime, or the completion of a therapeutic regime, to provide a base line result. This base-line can then be compared with the result obtained using identical methods either during or after such event. This information can be used for both diagnostic and prognostic purposes.

Kits

Another embodiment is directed to a kit containing a plurality of genes, preferably on a substrate. The kit also may comprise one or more containers or packages, along with reagents, solutions and possibly instructions for use.

All of the cited references are herein incorporated by reference. The invention is further described by the following Examples, which do not limit the invention in any manner.

EXAMPLES Example 1 Introduction to Study and Animal-Related Procedures

Introduction

Estrogen receptors are expressed in numerous organs, although only a few organs are considered classical targets for estrogens. A systematic survey of estrogen regulation of approximately 10,000 genes in 13 tissues from wild type and ERβKO mice treated subcutaneously with vehicle or 17β-estradiol (E2) for six weeks was conducted. As expected, the uterus and pituitary had the greatest number of genes regulated by E2, while, surprisingly, the kidney had the third largest number of regulated genes. Some of these kidney regulations may provide mechanisms for known physiological effects of estrogens. For example, E2 induction of CYP27B1, the rate limiting enzyme in the synthesis of 1,25-dihydroxy vitamin D, may explain the ability of estrogens to decrease urinary calcium excretion in women. In situ hybridizations localized E2 regulation in the kidney to the juxtamedullary proximal and distal collecting tubule epithelial cells in both the mouse and rat. E2 regulations in the kidney were intact in the ERβKO mice, and the ERα selective agonist propyl pyrazole triol acted similarly as E2, together suggesting an ERα mediated mechanism. Finally, the combination of the AF1-selective agonist tamoxifen plus mice expressing an AF1-deleted version of ERα (previously designated as ERα knockouts) allowed clear identification of genes dependent upon ERα AF1 activity and genes dependent upon ERα AF2 activity. Both AF1 and AF2 dependent genes were stimulated by E2 with the same ED₅₀, indicating that sensitivity of gene regulation in the kidney depends upon ER ligand binding and not on the subsequent ER activation mechanisms.

Animal-Related Procedure

Animals—Wldtype 129 strain female mice or Sprague-Dawley rats (bred at Wyeth or obtained from Taconic Farms) were placed on a casein-based diet at approximately 6 weeks of age. One week later, the animals were ovariectomized. Commencing the day after ovariectomy, each animal received a daily subcutaneous treatment with vehicle (50% DMSO, 50% phosphate buffered saline) or vehicle containing treatments for six weeks. Each group consisted of six or seven animals. Approximately 2 hours following the final treatment, the animals were euthanized with selected tissues frozen in liquid nitrogen for RNA analysis or on dry ice for histology.

Example 2 Preparation of Microarray

GeneChip—Total RNA was prepared separately from each individual organ by using Trizol (Invitrogen) followed by subsequent repurification on Rneasy columns (Qiagen). In general, two pools of RNA were created using equal amounts of RNA from three mice. For small organs such as pituitary, an equal amount of RNA from six animals was combined.

Target Preparation and Array Hybridization—Total RNA was used to generate biotin labeled cRNA target as described (8) which was hybridized to the murine MG_U74Av2 probe arrays (Affymetrix, Santa Clara, Calif.) for 16 h at 45° C. Eleven biotin-labeled cRNAs at defined concentration were spiked into each hybridization and used to convert average difference values to frequencies expressed as parts per million.

Example 3 Data Selection and Analysis

Pairwise comparisons were made between each of the treatments. We calculated the fold change ratio, the p-value based on Student's t-test, the number of present calls, and the expression level for each comparison. A confidence score (CS) was defined as CS(x)=FC(x)+PV(x)+PC(x)+EL(x) where FC, PV, PC and EL are scores assigned to the fold change, p-value, number of present calls, and the expression level, respectively. FC(x) was assigned 5 if the fold change ratio was greater then 1.95 and was assigned 0 if the ratio was between 1.95 and 1.5. PV(x) was assigned 3 if the p-value was less then 0.05 and was assigned 2 if the p-value was between 0.05 and 0.1. PC(x) was assigned 3 if at least 50% of the samples are called P by the Affymetrix algorithm and assigned 1 if only 25% of the samples are called P. EL(x) was assigned 3 if at least two samples had a frequency value of 20 or greater and assigned 1 if two samples only had a frequency greater then 15. Penalty points were assigned if the fold change was less then 1.5, the p-value was greater then 0.2 or the frequency values were below 15 ppm. CS(x) ranged for −14 to 14 with qualifiers having a score of 14 considered the most significant changes. Genes with 11 or more points in any one pairwise comparison were considered to be significant and were included for further analysis. Real-time PCR on individual RNA samples and histology analyses were performed essentially as described previously (9, 10).

Example 4 Discussion of Results

To begin a systematic survey of estrogen receptor regulation of gene expression in the mouse, ovariectomized wild-type (WT) and ERβKO mice were treated by daily subcutaneous administration of either vehicle or 20 μg/kg/day 17β-estradiol (E2) for six weeks. RNA prepared from 13 organs was analyzed by microarray for estrogen regulation of gene expression. The resulting data set was queried for genes whose regulation was dependent on ERα or ERβ. For ERα regulation, the basal expression level was predicted to be the same in WT and ERβKO mice, with E2 induction or suppression occurring in both WT and ERβKO mice. (FIG. 1). For ERβ regulation, basal expression was predicted to remain constant, with E2 induction or suppression occurring in WT mice but not in ERβKO mice. ERα pattern regulations were found in well known estrogen target tissues such as the uterus (514 inductions, 19 repressions), pituitary (56 inductions, 30 repressions) and bone marrow (3 inductions, 3 repressions). In contrast, essentially no genes could be discerned that fit the predicted ERβ regulation pattern in any tissue.

Surprisingly, the kidney had a very large number of genes regulated at least 2-fold by E2 (26 inductions, 4 repressions; FIG. 2). To further characterize E2 regulation of gene expression in the kidney, in situ hybridization was used to localize E2 induction of CYP7B1, TF, STAT5A and GADD45G In each case, induction of gene expression occurred in the juxtamedullary region of the kidney (FIG. 3) primarily in the proximal and distal tubule epithelium (not shown). Estrogen regulation of STAT5A and GADD45G also occurred in rat kidney juxtamedullary region (FIG. 4), demonstrating that the estrogen responsiveness of kidney is not limited to the mouse.

The ED₅₀s for E2 stimulation of CYP7B1, TF, STAT5A, and BCAT1 in the kidney were all very similar at about 3 μg/kg/day (FIG. 5). Although this is approximately 10-fold greater than the ED₅₀ dose of E2 required for uterine weight increases, the ED₅₀ for gene induction in the uterus can vary by 20-fold, from 0.2 ug/kg/day E2 for BCAT1 induction to 2.7 ug/kg/day for c-fos (FIG. 5). The E2 induction of gene expression in the kidney at the same dose as induction of well characterized genes such as c-fos in the uterus suggests that regulation of kidney gene expression occurs at physiological levels of E2.

Confirmation of the role of ERα in the induction of kidney gene expression was obtained with 4-propyl-1,3,5-Tris(4-hydroxy-phenyl)pyrazole (PPT) a compound which exclusively activates ERα but not ERβ (11). Treatment with PPT induced expression of CYP7B1, TF, STAT5A and BCAT1 to a similar extent as did treatment with E2 (FIG. 6). Further, two ERβ selective agonists (W-0292 and W-0070, both approximately 75-fold selective for ERβ compared to ERα by in vitro binding assays; data not shown) failed to stimulate expression of any of these four genes (FIG. 6). Finally, in agreement with previous results, ERα mRNA was detectable within the mouse kidney (FIG. 7). The regulation of these genes in WT and ERβKO mice, the similar E2 ED₅₀ for each gene, the activity of a selective ERα agonist, the inactivity of selective ERβ agonists, and the expression of ERα within the kidney together suggest a single, ERα mediated pathway for regulation of these genes.

It has been recognized that a commonly utilized strain of ERαKO mice (12) in fact expresses an ERα protein lacking only AF1, due to alternative splicings of the exon containing the targeted knockout mutation(13, 14). The resulting truncated ERα proteins, referred to here as ΔAF1-ERα, have the ability to stimulate expression of a synthetic estrogen response element driven promoter (14). As found previously for ERαKO mice, the level of this misspliced transcript in the uterus of ERαERβKO mice was lower than the level of full length message in WT mice (FIG. 7). Again as expected, the amount of intact ERα mRNA was much lower in the whole kidney than in uterus from WT mice. However, the level of ΔAF1-ERα mRNA was actually greater in the ERαERβKO kidney than was intact ERα mRNA in WT kidney. No ERβ mRNA could be detected in either uterus or kidney from the ERαERβKO mice.

The presence of ΔAF1-ERα at significant levels in the kidney allows determination of the relative contribution of AF1 and AF2 to E2 regulation of individual genes. To determine whether AF1 or AF2 regions of ERα were required for induction of CYP7B1, TF or BCAT1 in the kidney, WT, ERαERβKO or ERαKO mice were treated with E2 or the AF1 selective agonist tamoxifen (15). Expression of CYP7B1 was induced by E2 but not by tamoxifen in WT mice (FIG. 8), suggesting that induction of CYP7B1 occurred through AF2. Consistent with this hypothesis, E2 also increased CYP7B1 expression in mice expressing ΔAF1-ERα (either ERαERβKO or ERαKO mice). This E2 induction was blocked by an excess of ICI-182780, confirming the regulation occurred through ER. Together, these two lines of evidence suggest that induction of CYP7B1 is an AF2 dependent process. In contrast, TF expression was induced by both E2 and tamoxifen in WT mice. Neither compound could induce TF expression in ERαERβKO mice (which express only ΔAF1-ERα). This suggests that induction of TF occurs through an AF1 mediated pathway. Finally, BCAT1 was also induced by both E2 and tamoxifen in WT mice. However, in ERαERβKO mice, E2 stimulated BCAT1 expression but tamoxifen did not. These results suggest that BCAT1 expression can be stimulated through either AF1 or AF2 mechanisms. In WT mice, tamoxifen stimulates expression through AF1 only. Since ΔAF1-ERα lacks the AF1 region necessary for tamoxifen activity, tamoxifen cannot stimulate BCAT1 expression in ERαERβKO mice. In contrast, E2, which can stimulate BCAT1 expression through either AF1 or AF2, can still stimulate expression in the ΔAF1-ERα expressing mice.

Estrogen receptors α or β are found in almost all organs of the body, yet relatively few tissues are considered targets for estrogen action. To begin to develop a more complete understanding of estrogen biology, estrogen responsive genes in 13 tissues from WT and ERβKO mice were characterized. In general, many tissues showed patterns of E2 regulation consistent with an ERα mechanism, including such known target organs as uterus, pituitary, and bone. Surprisingly, no E2 regulations were found that fit the expected pattern for ERβ regulations. This was true even in organs expressing moderately high levels of ERβ such as the bladder and lung (7). At least three mechanisms could explain the lack of detection of expected ERβ responses. First, it has been proposed that a major function of ERβ is to modulate the activity of ERα(16). For example, expression of the Ki-67 protein was constitutively elevated in uterus of ERβKO mice, i.e. in the ERβKO mice its expression was always equivalent to the E2 stimulated levels in WT animals (17). The survey criteria used here would not detect this pattern. Further analysis of these data has revealed many genes in multiple tissues which also have this “nonclassical” pattern of regulation whereby expression is constitutively elevated in both vehicle and E2 treated ERβKO mice (data not shown). Second, analysis of whole organs may easily miss regulations occurring in only selected cell subtypes within an organ. For example, initial analysis of kidney did not identify GADD45G as being regulated by E2, because GADD45G expression is regulated only in tubule epithelial cells. The unregulated expression of GADD45G throughout most of the kidney sufficiently diminished the fold induction so as to be less than 2-fold in whole organ samples. The combination of laser capture microdissection with microarray technology (18) should allow detection of ERβ regulated genes with a classical pattern of regulation.

This global survey demonstrates that, unexpectedly, the kidney had a very large number of regulated genes. Both genetic approaches (FIG. 2) and pharmacological approaches (FIG. 6) demonstrated that E2 regulation in the kidney was mediated through ERα. Expression of CYP7B1, TF, STAT5A, and even genes such as GADD45G which are expressed throughout the kidney showed regulation only in tubule epithelium (FIG. 3). Additionally, KIM-1, the rat counterpart of mouse TIM1 and TIM2 (20) is also expressed in proximal tubule epithelial cells (21). Finally, ³H-E2 binding localizes to proximal tubule cells following administration to rats(22). Together, these results suggest that ERα directly regulates gene expression in tubule epithelial cells.

Although the observed regulations in the kidney were mediated by ERα, the mechanism of activation of gene expression by ERα was gene specific. Thus studies using tamoxifen, which activates ERα through AF1, along with studies using ΔAF1-ERαKO mice (previously designated as ERKO mice) together indicate that E2 induction of CYP7B1 expression occurred predominantly through an AF1-dependent mechanism, E2 induction of TF expression occurred predominantly through an AF2-dependent mechanism, and E2 induction of BCAT1 expression occurred through both AF1 and AF2 mechanisms (FIG. 8). The ED₅₀ values for E2 stimulation of these three genes were all very similar (FIG. 5). Thus, whether a gene is induced through either AF1 or AF2 mechanism does not influence the sensitivity of the gene in the kidney to plasma estrogen levels. Rather, the data indicate that the binding of E2 to ERα would be the rate limiting step in induction of gene expression in the kidney. The maximal fold regulation varied between genes and may depend upon whether and AF1 or AF2 dependent pathway is utilized.

Analysis of 10 kb of upstream putative promoter sequences of E2 induced genes identified good matches to the consensus estrogen response element (ERE) in only a few genes, although ERE half-sites could be identified in most promoters. Many of these genes may be activated through nonclassical ERα mechanisms such as the combination of an ERE half-site with Sp1 binding sites (41). It is unlikely that a nonclassical ERα/AP1 stimulatory mechanism is responsible for these regulations, since ICI182780 functions as a partial agonist in this mechanism (42) while ICI182780 was a complete antagonist for E2 regulation of gene expression in the kidney (FIG. 8). Additionally, E2 induced expression of the transcription factors PHD2, ELF3, STAT5A and STAT5B. It is possible that E2 induction of these transcription factors resulted in the subsequent increase in expression of the remaining genes. For example, CIS2 is a known target for induction by STAT transcription factors (43), suggesting that the E2 induction of CIS2 is mediated indirectly through the E2 induction of STAT5A and STAT5B. TABLE II Genes Regulated By Estrogen in Kidney, Uterus and Pituitary Gland Unigene Code Full name Why Kidney Tissue Factor Mm.3742 Coagulation factor III Mechanism is ERα AF1 dependent CYP7B1 Mm.6216 Cytochrome P450, 40 (25-hydroxyvitamin D3 1 alpha- Mechanism is ERα AF2 dependent hydroxylase) BCAT1 Mm.4606 Branched chain aminotransferase 1, cytosolic Mechanism is ERα AF1 + AF2 dependent STAT5A Mm.4697 Signal transducer and activator of transcription 5A Regulated in multiple species (mouse and rat) GADD45G Mm.9653 Growth arrest and DNA-damage-inducible 45 gamma Regulated in multiple species (mouse and rat) BHMT Mm.21983 Betaine-homocysteine methyltransferase A repression by estrogens SAHH Mm.2573 S-adenosylhomocysteine hydrolase NTT73 Mm.4327 SODIUM- AND CHLORIDE-DEPENDENT TRANSPORTER NTT73 ABCC3 Mm.23942 ATP-binding cassette, sub-family C (CFTR/MRP), member 3 Uterus SFRP4 Mm.42095 Secreted frizzled-related sequence protein 4 Induced by estrogens in mouse uterus and human endometrium Deiodinase, type II Mm.21389 Deiodinase, iodothyronine, type II Induced by estrogens in mouse uterus and human endometrium Procollagen, type I, Mm.22621 Procollagen, type I, alpha 1 Induced by estrogens in mouse uterus and alpha 1 human endometrium vimentin Mm.7 Vimentin Induced by estrogens in mouse uterus and human endometrium IGFBP4 Mm.22248 Insulin-like growth factor binding protein 4 Induced by estrogens in mouse uterus and human endometrium Scavenger receptor Mm.1227 Macrophage scavenger receptor 1 Repressed by estrogens in mouse uterus and human endometrium AI121305 Mm.29959 RIKEN cDNA 1600029D21 a set of genes induced by estrogens with a range of ED50 values ALOX15 Mm.4584 Arachidonate 15-lipoxygenase a set of genes induced by estrogens with a range of ED50 values BCAT1 Mm.4606 Branched chain aminotransferase 1, cytosolic a set of genes induced by estrogens with a range of ED50 values SIAMOX Mm.7190 Amiloride binding protein 1 (amine oxidase, copper- a set of genes induced by estrogens with a containing) range of ED50 values C3 Mm.19131 Complement component 3 a set of genes induced by estrogens with a range of ED50 values FOS Mm.5043 FBJ osteosarcoma oncogene a set of genes induced by estrogens with a range of ED50 values MAP2k1 Mm.1059 Mitogen activated protein kinase kinase 1 a set of genes induced by estrogens with a range of ED50 values CEBPb Mm.4863 CCAAT/enhancer binding protein (C/EBP), beta a set of genes induced by estrogens with a range of ED50 values EGR1 Mm.181959 Early growth response 1 a set of genes induced by estrogens with a range of ED50 values CYP1A1 Mm.14089 Cytochrome P450, 1a1, aromatic compound inducible Repressed by estrogens Pituitary STAT5B Mm.34064 Signal transducer and activator of transcription 5B Induced by estrogens GADD45G Mm.9653 Growth arrest and DNA-damage-inducible 45 gamma Induced by estrogens Kallikrein-9 Mm.200410 Kallikrein 9 Induced by 17b-estradiol, not by Premarin FSHb Mm.46711 Follicle stimulating hormone beta Repressed by estrogens

TABLE III Genes Regulated By Estrogen in the Uterus Mousedata. Mean WT E2 Qualifier Pub_Name Gene Name Tissue Fold Change 94120_s_at SPRR2F small proline-rich protein 2F Uterus 38.71 97413_at UNK_AI121305 ESTs, Weakly similar to AF189262_1 GABA-A receptor epsilon Uterus 31.68 like subunit [R. norvegicus] 101130_at COLA2 procollagen, type I, alpha 2 Uterus 29.57 103526_at PDI2 peptidyl arginine deiminase, type II Uterus 23.01 99059_at ELF3 E74-like factor 3 Uterus 22.03 95343_at PDI1 peptidyl arginine deiminase, type I Uterus 21.72 101115_at LTF lactotransferrin Uterus 19.01 93481_at UNK_AI846720 Cluster Incl AI846720: UI-M-AN1-afi-h-09-0-UI.s1 Mus Uterus 17.96 musculus cDNA, 3′ end/clone = UI-M-AN1-afi-h-09-0-UI/ clone_end = 3′/gb = AI846720/gi = 5490626/ug = Mm.7124/ len = 161/STRA = for 93097_at ARG1 arginase 1, liver Uterus 17.45 104249_g_at UNK_AW227650 ESTs, Highly similar to TRANSLOCON-ASSOCIATED Uterus 16.87 PROTEIN, GAMMA SUBUNIT [Rattus norvegicus] 93797_g_at LDH1 lactate dehydrogenase 1, A chain Uterus 16.5 101761_f_at SPRR2C small proline-rich protein 2C Uterus 16.48 102805_at CEACAM1 CEA-related cell adhesion molecule 1 Uterus 16 98064_at GLYCAM1 glycosylation dependent cell adhesion molecule 1 Uterus 15.46 AFFX- GAPDH5_Mm_AFFX Glyceraldehyde-3-phospate dehydrogenase 5′ control Uterus 15.2 GapdhMur/ sequence (M. musculus) [AFFX] M32599_5_(—) at AFFX- GAPDH5_Mm_AFFX Glyceraldehyde-3-phospate dehydrogenase 5′ control Uterus 15.2 GapdhMur/ sequence (M. musculus) [AFFX] M32599_5_(—) at AFFX- GAPDH5_Mm_AFFX Glyceraldehyde-3-phospate dehydrogenase 5′ control Uterus 15.2 GapdhMur/ sequence (M. musculus) [AFFX] M32599_5_(—) at 96605_at UNK_AI787183 ESTs, Weakly similar to AF1154269_1 LR8 [M. musculus] Uterus 14.98 102806_g_at CEACAM1 CEA-related cell adhesion molecule 1 Uterus 14.48 93860_i_at UNK_M17327 Mouse endogenous murine leukemia virus modified polytropic Uterus 13.83 provirus DNA, complete cds 101707_at ALDH1A7 alcohol dehydrogenase family 1, subfamily A7 Uterus 13.63 104486_at UNK_AI850558 ESTs, Highly similar to ALPHA-2-MACROGLOBULIN Uterus 13.29 PRECURSOR [Homo sapiens] 98423_at GJB2 gap junction membrane channel protein beta 2 Uterus 12.6 104182_at HGFAC hepatocyte growth factor activator Uterus 12 94789_r_at TUBB5 tubulin, beta 5 Uterus 11.55 98822_at ISG15 interferon-stimulated protein (15 kDa) Uterus 11.5 97826_at UNK_AI465965 ESTs, Weakly similar to IgG Fc binding protein [M. musculus] Uterus 10.95 100026_at BCAT1 branched chain aminotransferase 1, cytosolic Uterus 10.35 102316_at CAPN5 calpain 5 Uterus 10.01 98092_at D5WSU111E DNA segment, Chr 5, Wayne State University 111, expressed Uterus 9.77 102918_at MUC1 mucin 1, transmembrane Uterus 9.74 97173_f_at H2-K2 histocompatibility 2, K region locus 2 Uterus 9.71 93497_at C3 complement component 3 Uterus 9.63 93517_at COL6A2 procollagen, type VI, alpha 2 Uterus 9.57 92796_at AKP2 alkaline phosphatase 2, liver Uterus 9.36 103824_at WFS1 Wolfram syndrome 1 homolog (human) Uterus 9.15 99378_f_at UNK_M18837 Mouse MHC class I Q4 beta-2-microglobulin (Qb-1) gene, Uterus 8.9 complete cds 99561_f_at CLDN7 claudin 7 Uterus 8.84 94305_at COLA1 procollagen, type I, alpha 1 Uterus 8.78 92223_at C1QC complement component 1, q subcomponent, c polypeptide Uterus 8.63 100134_at ENG endoglin Uterus 8.53 92550_at KRT1-19 keratin complex 1, acidic, gene 19 Uterus 8.53 103905_at UNK_AI314958 ESTs, Highly similar to CARBONIC ANHYDRASE VI [Ovisaries] Uterus 8.44 93285_at UNK_AI845584 ESTs, Highly similar to DUS6_RAT DUAL SPECIFICITY Uterus 8.39 PROTEIN PHOSPHATASE 6 [R. norvegicus] 92777_at CYR61 cysteine rich protein 61 Uterus 8.3 97819_at GSTTL-PENDING glutathione S-transferase like Uterus 8.12 93479_at UNK_AW122413 Cluster Incl AW122413: UI-M-BH2.2-aow-f-03-0-UI.s1 Mus Uterus 7.99 musculus cDNA, 3′ end/clone = UI-M-BH2.2-aow-f-03-0-UI/ clone_end = 3′/gb = AW122413/gi = 6097916/ug = Mm.7113/ len = 470/STRA = rev 104099_at PGLYRP peptidoglycan recognition protein Uterus 7.98 97507_at PPICAP peptidylprolyl isomerase C-associated protein Uterus 7.86 94876_f_at UNK_AI849207 ESTs, Weakly similar to AF218940_1 formin-2 [M. musculus] Uterus 7.77 96911_at GNB2 guanine nucleotide binding protein, beta 2 Uterus 7.56 92608_at CSRP cysteine rich protein Uterus 7.56 94269_at RABAC1 Rab acceptor 1 (prenylated) Uterus 7.46 93861_f_at UNK_M17327 Mouse endogenous murine leukemia virus modified polytropic Uterus 7.39 provirus DNA, complete cds 101110_at COL6A3 procollagen, type VI, alpha 3 Uterus 7.37 93974_at 33 POLYPEPTIDE□ ESTs, Highly similar to G33_RAT GENE 33 POLYPEPTIDE□ Uterus 7.34 [R. NORVEGICUS] [R. norvegicus] 98758_at ALOX15 arachidonate 15-lipoxygenase Uterus 7.33 99379_f_at UNK_M27034 Mouse MHC class I D-region cell surface antigen (D2d) gene, Uterus 7.23 complete cds 93078_at LY6 lymphocyte antigen 6 complex Uterus 7.17 93290_at PNP purine-nucleoside phosphorylase Uterus 7.12 101979_at GADD45G growth arrest and DNA-damage-inducible 45 gamma Uterus 7.06 99452_at LISCH7-PENDING liver-specific bHLH-Zip transcription factor Uterus 7 94274_at PFDN5 prefoldin 5 Uterus 6.97 92880_at MFGE8 milk fat globule-EGF factor 8 protein Uterus 6.95 101294_g_at G6PD2 glucose-6-phosphate dehydrogenase 2 Uterus 6.95 92759_at LAMB3 laminin, beta 3 Uterus 6.88 92585_at MAP2K1 mitogen activated protein kinase kinase 1 Uterus 6.86 95232_at HNRPL heterogeneous nuclear ribonucleoprotein L Uterus 6.79 104410_at MIDN-PENDING midnolin Uterus 6.75 96075_at WDR1 WD repeat domain 1 Uterus 6.71 95631_at PPP4C protein phosphatase 4, catalytic subunit Uterus 6.68 100412_g_at AEBP1 AE-binding protein 1 Uterus 6.67 96634_at UNK_AI850090 ESTs, Weakly similar to cDNA EST EMBL: C07816 comes from Uterus 6.65 this gene [C. elegans] 97282_at MELA melanoma antigen, 80 kDa Uterus 6.6 98511_at RALY hnRNP-associated with lethal yellow Uterus 6.55 94199_at KAP kidney androgen regulated protein Uterus 6.45 93818_g_at RNP24-PENDING coated vesicle membrane protein Uterus 6.32 98331_at COL3A1 procollagen, type III, alpha 1 Uterus 6.29 92642_at CAR2 carbonic anhydrase 2 Uterus 6.28 103278_at PDI4 peptidyl arginine deiminase, type IV Uterus 6.23 101542_f_at DDX3 DEAD (aspartate-glutamate-alanine-aspartate) box polypeptide 3 Uterus 6.23 93793_at LASP1 LIM and SH3 protein 1 Uterus 6.17 94817_at SERPINH1 serine (or cysteine) proteinase inhibitor, clade H (heat shock Uterus 6.06 protein 47), member 1 99569_at KRT2-18 keratin complex 2, basic, gene 18 Uterus 6.04 95705_s_at ACTX melanoma X-actin Uterus 5.94 92368_at RAMP3 receptor (calcitonin) activity modifying protein 3 Uterus 5.93 102292_at GADD45A growth arrest and DNA-damage-inducible 45 alpha Uterus 5.93 94384_at IER3 immediate early response 3 Uterus 5.84 103438_at DIO2 deiodinase, iodothyronine, type II Uterus 5.83 97882_at SEC61A SEC61, alpha subunit (S. cerevisiae) Uterus 5.81 93574_at PEDF pigment epithelium-derived factor Uterus 5.81 99622_at KLF4 Kruppel-like factor 4 (gut) Uterus 5.74 100981_at IFIT1 interferon-induced protein with tetratricopeptide repeats 1 Uterus 5.74 99645_at UNK_AW048484 Cluster Incl AW048484: UI-M-BH1-alj-d-10-0-UI.s1 Mus Uterus 5.67 musculus cDNA, 3′ end/clone = UI-M-BH1-alj-d-10-0-UI/ clone_end = 3′/gb = AW048484/gi = 5909018/ug = Mm.43640/ len = 458/STRA = for 95444_at UNK_AW122274 ESTs, Weakly similar to CG1534 gene product Uterus 5.67 [D. melanogaster] 99931_at LAMA5 laminin, alpha 5 Uterus 5.66 100130_at JUN Jun oncogene Uterus 5.64 100618_f_at SLC25A5 solute carrier family 25 (mitochondrial carrier; adenine Uterus 5.64 nucleotide translocator), member 5 101929_at UNK_AI836322 Cluster Incl AI836322: UI-M-AQ0-aag-a-02-0-UI.s2 Mus Uterus 5.63 musculus cDNA, 3′ end/clone = UI-M-AQ0-aag-a-02-0-UI/ clone_end = 3′/gb = AI836322/gi = 5470530/ug = Mm.939/ len = 211/STRA = for 100609_at UNK_AF049850 Cluster Incl AF049850: Mus musculus major histocompatibility Uterus 5.58 locus class III region-complement C4 (C4) and cytochrome P450 hydroxylase A (CYP21OH-A) genes, complete cds; slp pseudogene, complete sequence; NG6, SKI, and complement factor B (Bf) genes, comp 95637_at UNK_AI838592 ESTs, Moderately similar to ENDOTHELIAL ACTIN-BINDING Uterus 5.46 PROTEIN [Homo sapiens] 101367_at DCTN1 dynactin 1 Uterus 5.42 101681_f_at H2-BL histocompatibility 2, blastocyst Uterus 5.24 100557_g_at UNK_AW121930 ESTs, Highly similar to EUKARYOTIC INITIATION FACTOR Uterus 5.21 4B [Homo sapiens] 93985_at UNK_AW120868 ESTs, Highly similar to hypothetical protein [H. sapiens] Uterus 5.18 92851_at CP ceruloplasmin Uterus 5.14 99109_at IER2 immediate early response 2 Uterus 5.13 99632_at MAD2L1 MAD2 (mitotic arrest deficient, homolog)-like 1 (yeast) Uterus 5.13 94307_at FBLN1 fibulin 1 Uterus 5.1 92232_at CISH3 cytokine inducible SH2-containing protein 3 Uterus 5.09 92611_at GPIAP-PENDING GPI-anchored membrane protein 1 Uterus 5.07 104333_at D17H6S56E-5 DNA segment, Chr 17, human D6S56E 5 Uterus 5.07 101016_at ARF1 ADP-ribosylation factor 1 Uterus 5.06 103460_at UNK_AI849939 ESTs, Moderately similar to unnamed protein product Uterus 5.05 [H. sapiens] 94309_g_at FBLN1 fibulin 1 Uterus 4.95 99927_at CFI complement component factor I Uterus 4.94 96278_at UNK_AI846553 ESTs, Weakly similar to DIA1_MOUSE DIAPHANOUS Uterus 4.84 PROTEIN HOMOLOG 1 [M. musculus] 103345_at UNK_AW046708 ESTs, Highly similar to SPECTRIN ALPHA CHAIN, NON- Uterus 4.83 ERYTHROID [Rattus norvegicus] 95794_f_at SPRR2I small proline-rich protein 2I Uterus 4.83 101908_s_at CEACAM2 CEA-related cell adhesion molecule 2 Uterus 4.8 104144_at GTPBP2 GTP binding protein 2 Uterus 4.8 102362_i_at JUNB Jun-B oncogene Uterus 4.79 AFFX- GAPDHM_Mm_AFFX Glyceraldehyde-3-phospate dehydrogenase middle control Uterus 4.79 GapdhMur/ sequence (M. musculus) [AFFX] M32599_M_at AFFX- GAPDHM_Mm_AFFX Glyceraldehyde-3-phospate dehydrogenase middle control Uterus 4.79 GapdhMur/ sequence (M. musculus) [AFFX] M32599_M_at AFFX- GAPDHM_Mm_AFFX Glyceraldehyde-3-phospate dehydrogenase middle control Uterus 4.79 GapdhMur/ sequence (M. musculus) [AFFX] M32599_M_at 94246_at ETS2 E26 avian leukemia oncogene 2, 3′ domain Uterus 4.78 98930_at COPE coatomer protein complex, subunit epsilon Uterus 4.76 98928_at CORO1B coronin, actin binding protein 1B Uterus 4.76 94821_at XBP1 X-box binding protein 1 Uterus 4.69 95708_at D3UCLA1 DNA segment, Chr 3, University of California at Los Angeles 1 Uterus 4.66 96284_at UNK_AW121446 ESTs, Moderately similar to CASEIN KINASE I, GAMMA Uterus 4.64 ISOFORM [Bos taurus] 104279_at UNK_AW125116 ESTs, Highly similar to DNA-DIRECTED RNA POLYMERASE Uterus 4.62 II 14.4 KD POLYPEPTIDE [Homo sapiens; Cricetulus griseus] 93541_at TAGLN transgelin Uterus 4.62 93798_at LDH1 lactate dehydrogenase 1, A chain Uterus 4.61 99926_at PIGR polymeric immunoglobulin receptor Uterus 4.61 99338_at UNK_AA674798 ESTs, Highly similar to TIP120 [R. norvegicus] Uterus 4.6 93066_at GRN granulin Uterus 4.6 99366_at UNK_AI553536 Cluster Incl AI553536: vw39e06.x1 Mus musculus cDNA, 3′ end/ Uterus 4.58 clone = IMAGE-1246210/clone_end = 3′/gb = AI553536/ gi = 4485899/ug = Mm.5675/len = 408/STRA = rev 103556_at UNK_AI840158 Cluster Incl AI840158: UI-M-AO0-acc-d-08-0-UI.s1 Mus Uterus 4.55 musculus cDNA, 3′ end/clone = UI-M-AO0-acc-d-08-0-UI/ clone_end = 3′/gb = AI840158/gi = 5474371/ug = Mm.19081/ len = 406/STRA = for 101095_at MFAP2 microfibrillar-associated protein 2 Uterus 4.53 100323_at AMD2 S-adenosylmethionine decarboxylase 2 Uterus 4.5 102161_f_at H2-Q2 histocompatibility 2, Q region locus 2 Uterus 4.5 101955_at HSPA5 heat shock 70 kD protein 5 (glucose-regulated protein, 78 kD) Uterus 4.49 95654_at UNK_AF109905 Cluster Incl AF109905: Mus musculus major histocompatibility Uterus 4.49 locus class III regions Hsc70t gene, partial cds; smRNP, G7A, NG23, MutS homolog, CLCP, NG24, NG25, and NG26 genes, complete cds; and unknown genes/cds = (0,725)/gb = AF109905/ gi = 3986751/ug = Mm.29 98107_at UNK_AW123801 Cluster Incl AW123801: UI-M-BH2.1-apm-e-08-0-UI.s1 Mus Uterus 4.48 musculus cDNA, 3′ end/clone = UI-M-BH2.1-apm-e-08-0-UI/ clone_end = 3′/gb = AW123801/gi = 6099331/ug = Mm.34796/ len = 367/STRA = for 92925_at CEBPB CCAAT/enhancer binding protein (C/EBP), beta Uterus 4.47 92625_at NME2 expressed in non-metastatic cells 2, protein (NM23B) Uterus 4.46 (nucleoside diphosphate kinase) 96283_at ITM3-PENDING integral membrane protein 3 Uterus 4.43 97809_at UNK_AF109906 Cluster Incl AF109906: Mus musculus MHC class III region RD Uterus 4.4 gene, partial cds; Bf, C2, G9A, NG22, G9, HSP70, HSP70, HSC70t, and smRNP genes, complete cds; G7A gene, partial cds; and unknown genes/cds = (0,3002)/gb = AF109906/ gi = 3986763/ug = Mm.28155/len = 300 103709_at UNK_AA763466 Cluster Incl AA763466: vw54f05.r1 Mus musculus cDNA, 5′ end/ Uterus 4.37 clone = IMAGE-1247649/clone_end = 5′/gb = AA763466/ gi = 2813213/ug = Mm.24093/len = 379/STRA = for 98562_at C1QA complement component 1, q subcomponent, alpha polypeptide Uterus 4.37 92644_s_at MYB myeloblastosis oncogene Uterus 4.37 99624_at RPL5 ribosomal protein L5 Uterus 4.33 104093_at LSP1 lymphocyte specific 1 Uterus 4.31 99942_s_at CNN1 calponin 1 Uterus 4.31 101055_at PPGB protective protein for beta-galactosidase Uterus 4.3 100059_at CYBA cytochrome b-245, alpha polypeptide Uterus 4.29 94868_at UNK_AW049812 ESTs, Highly similar to GLUTAMINYL-TRNA SYNTHETASE Uterus 4.28 [Homo sapiens] 93751_at UNK_AW048157 ESTs, Highly similar to PROBABLE UBIQUITIN CARBOXYL- Uterus 4.27 TERMINAL HYDROLASE [Mus musculus] 101061_at UNK_AI845293 ESTs, Highly similar to TRANSLOCON-ASSOCIATED Uterus 4.26 PROTEIN, BETA SUBUNIT PRECURSOR [Homo sapiens] 101916_at DHCR7 7-dehydrocholesterol reductase Uterus 4.25 93327_at UNK_AI842665 ESTs, Highly similar to HYPOTHETICAL 13.5 KD PROTEIN Uterus 4.25 C45G9.7 IN CHROMOSOME III [Caenorhabditis elegans] 102968_at GGTLA1 gamma-glutamyltransferase-like activity 1 Uterus 4.24 99106_at COPS6 COP9 (constitutive photomorphogenic), subunit 6 (Arabidopsis) Uterus 4.22 97160_at SPARC secreted acidic cysteine rich glycoprotein Uterus 4.22 96943_at UNK_AW125234 ESTs, Highly similar to FUSCA PROTEIN FUS6 [Arabidopsis Uterus 4.2 thaliana] 97320_at UNK_AI842734 ESTs, Weakly similar to KE4_MOUSE HISTIDINE-RICH Uterus 4.18 PROTEIN KE4□ [M. musculus] 96353_at UNK_AW125346 ESTs, Moderately similar to AF151028_1 HSPC194 Uterus 4.17 [H. sapiens] 94854_g_at GNB1 guanine nucleotide binding protein, beta 1 Uterus 4.15 97894_at UNK_AF109905 Cluster Incl AF109905: Mus musculus major histocompatibility Uterus 4.14 locus class III regions Hsc70t gene, partial cds; smRNP, G7A, NG23, MutS homolog, CLCP, NG24, NG25, and NG26 genes, complete cds; and unknown genes/cds = (0,3791)/ gb = AF109905/gi = 3986751/ug = Mm.2 93390_g_at PROM prominin Uterus 4.13 103429_i_at UNK_AW125330 ESTs, Moderately similar to unnamed protein product Uterus 4.1 [H. sapiens] 96186_at UNK_AI839286 ESTs, Moderately similar to Unknown [H. sapiens] Uterus 4.09 103335_at LGALS9 lectin, galactose binding, soluble 9 Uterus 4.07 101393_at ANXA3 annexin A3 Uterus 4.07 93389_at PROM prominin Uterus 4.06 103888_at RBPMS RNA-blnding protein gene with multiple splicing Uterus 4.06 96258_at D13ERTD372E DNA segment, Chr 13, ERATO Doi 372, expressed Uterus 4.03 95161_at D10ERTD73E DNA segment, Chr 10, ERATO Doi 73, expressed Uterus 4.03 96869_at GABARAP gamma-aminobutyric acid receptor associated protein Uterus 4.02 101558_s_at PSMB5 proteasome (prosome, macropain) subunit, beta type 5 Uterus 4 96883_at EIF3S4 eukaryotic translation initiation factor 3, subunit 4 (delta, 44 kDa) Uterus 3.98 99549_at OGN osteoglycin Uterus 3.95 101781_f_at UNK_V00754 HISTONE H3.4 Uterus 3.95 93975_at 33 POLYPEPTIDE□ ESTs, Highly similar to G33_RAT GENE 33 POLYPEPTIDE□ Uterus 3.91 [R. NORVEGICUS] [R. norvegicus] 92930_at DLX5 distal-less homeobox 5 Uterus 3.91 95462_at UNK_AW060951 ESTs, Highly similar to unknown [R. norvegicus] Uterus 3.9 102791_at PSMB8 proteosome (prosome, macropain) subunit, beta type 8 (large Uterus 3.89 multifunctional protease 7) 95215_f_at UBC ubiquitin C Uterus 3.89 92850_at UNK_AI836446 ESTs, Moderately similar to KIAA1398 protein [H. sapiens] Uterus 3.88 100332_s_at PRDX5-RS3 peroxiredoxin 5, related sequence 3 Uterus 3.87 100561_at IQGAP1 IQ motif containing GTPase activating protein 1 Uterus 3.84 98446_s_at EPHB4 Eph receptor B4 Uterus 3.82 100771_at LY57 lymphocyte antigen 57 Uterus 3.81 103547_at UNK_AI837116 Cluster Incl AI837116: UI-M-AK0-adc-e-09-0-UI.s1 Mus Uterus 3.81 musculus cDNA, 3′ end/clone = UI-M-AK0-adc-e-09-0-UI/ clone_end = 3′/gb = AI837116/gi = 5471329/ug = Mm.23723/ len = 323/STRA = rev 104365_at SCAMP2 secretory carrier membrane protein 2 Uterus 3.8 93496_at UNK_AI852098 ESTs, Weakly similar to AF104033_1 MUEL protein Uterus 3.79 [M. musculus] 100970_at AKT thymoma viral proto-oncogene Uterus 3.79 96318_at D17WSU104E DNA segment, Chr 17, Wayne State University 104, expressed Uterus 3.78 93430_at CMKOR1 chemokine orphan receptor 1 Uterus 3.75 92882_at RAB1 RAB1, member RAS oncogene family Uterus 3.74 97824_at D11ERTD175E DNA segment, Chr 11, ERATO Doi 175, expressed Uterus 3.72 99991_at IL17R interleukin 17 receptor Uterus 3.72 100684_at PRKCSH protein kinase C substrate 80K-H Uterus 3.72 96935_at UNK_AW011791 ESTs, Moderately similar to epithelial protein up-regulated in Uterus 3.71 carcinoma [H. sapiens] 93500_at ALAS1 aminolevulinic acid synthase 1 Uterus 3.69 100554_at PDLIM1 PDZ and LIM domain 1 (elfin) Uterus 3.67 94105_at CDC42 cell division cycle 42 homolog (S. cerevisiae) Uterus 3.66 101486_at PSMB10 proteasome (prosome, macropain) subunit, beta type 10 Uterus 3.66 96155_at UNK_AW049359 ESTs, Highly similar to AF177476_1 CDK5 activator-binding Uterus 3.65 protein [R. norvegicus] 99475_at CISH2 cytokine inducible SH2-containing protein 2 Uterus 3.64 102767_at AA536815 EST AA536815 Uterus 3.64 104315_at UNK_AI846773 Cluster Incl AI846773: UI-M-AO1-ael-f-02-0-UI.s1 Mus Uterus 3.64 musculus cDNA, 3′ end/clone = UI-M-AO1-ael-f-02-0-UI/ clone_end = 3′/gb = AI846773/gi = 5490679/ug = Mm.22413/ len = 322/STRA = for 104389_at UNK_AW049360 ESTs, Weakly similar to T17295 hypothetical protein Uterus 3.63 DKFZp434H132.1 - human [H. sapiens] 93833_s_at UNK_X05862 Cluster Incl X05862: Mouse H2B and H2A histone genes Uterus 3.61 (291A)/cds = (0,380)/gb = X05862/gi = 51302/ug = Mm.21579 /len = 381/STRA = for 101881_g_at COL18A1 procollagen, type XVIII, alpha 1 Uterus 3.61 100569_at ANXA2 annexin A2 Uterus 3.6 94561_at UNK_AI836140 Mus musculus epithelial protein lost in neoplasm-a (Eplin) Uterus 3.59 mRNA, complete cds 95608_at CTSB cathepsin B Uterus 3.57 96709_at UNK_AI839839 ESTs, Highly similar to EST00098 protein [H. sapiens] Uterus 3.57 93102_f_at ACTG2 actin, gamma 2, smooth muscle, enteric Uterus 3.55 99477_at GNG12 guanine nucleotide binding protein (G protein), gamma 12 Uterus 3.55 94237_at D6WSU137E DNA segment, Chr 6, Wayne State University 137, expressed Uterus 3.55 98937_at TBRG1 transforming growth factor beta regulated gene 1 Uterus 3.53 94503_at UNK_AI842492 ESTs, Highly similar to RAS-RELATED PROTEIN RAB-8 Uterus 3.5 [Homo sapiens; Canis familiaris] 99019_at POR P450 (cytochrome) oxidoreductase Uterus 3.49 104623_at TLE3 transducin-like enhancer of split 3, homolog of Drosophila Uterus 3.47 E(spl) 92866_at H2-AA histocompatibility 2, class II antigen A, alpha Uterus 3.46 103200_at UNK_AA711773 Cluster Incl AA711773: vu58g05.r1 Mus musculus cDNA, 5′ end/ Uterus 3.44 clone = IMAGE-1195640/clone_end = 5′/gb = AA711773/ gi = 2721691/ug = Mm.1902/len = 473/STRA = for 104100_at UNK_AI845915 Cluster Incl AI845915: UI-M-AK1-aex-d-02-0-UI.s1 Mus Uterus 3.42 musculus cDNA, 3′ end/clone = UI-M-AK1-aex-d-02-0-UI/ clone_end = 3′/gb = AI845915/gi = 5489821/ug = Mm.21864/ len = 208/STRA = for 94063_at SEMA4A sema domain, immunoglobulin domain (Ig), transmembrane Uterus 3.42 domain (TM) and short cytoplasmic domain, (semaphorin) 4A 95752_at UNK_AI837369 ESTs, Highly similar to unnamed protein product [H. sapiens] Uterus 3.42 97409_at IFI1 interferon inducible protein 1 Uterus 3.41 95749_at UNK_AW122364 ESTs, Highly similar to ARGR_HUMAN ARGININE-RICH Uterus 3.41 PROTEIN□ [H. sapiens] 104110_at UNK_AW060515 Cluster Incl AW060515: UI-M-BH1-ann-d-07-0-UI.s1 Mus Uterus 3.39 musculus cDNA, 3′ end/clone = UI-M-BH1-ann-d-07-0-UI/ clone_end = 3′/gb = AW060515/gi = 6008266/ug = Mm.21919/ len = 330/STRA = for 99101_at EIF3S7 eIF3 p66 Uterus 3.39 94966_at G6PDX glucose-6-phosphate dehydrogenase X-linked Uterus 3.38 92567_at COL5A2 procollagen, type V, alpha 2 Uterus 3.37 103016_s_at CD68 CD68 antigen Uterus 3.36 AFFX-b- BACTIN3_Mm_AFFX Beta-actin 3′ control sequence (M. musculus) [AFFX] Uterus 3.35 ActinMur/M 12481_3_at AFFX-b- BACTIN3_Mm_AFFX Beta-actin 3′ control sequence (M. musculus) [AFFX] Uterus 3.35 ActinMur/M 12481_3_at AFFX-b- BACTIN3_Mm_AFFX Beta-actin 3′ control sequence (M. musculus) [AFFX] Uterus 3.35 ActinMur/M 12481_3_at 96653_at APP amyloid beta (A4) precursor protein Uterus 3.35 99872_s_at FTL1 ferritin light chain 1 Uterus 3.34 97125_f_at LOC56628 MHC (A.CA/J(H-2K-f) class I antigen Uterus 3.33 94288_at HIS1A histone H1 Uterus 3.32 93276_at HN1 hematological and neurological expressed sequence 1 Uterus 3.29 93071_at TIF1B transcriptional intermediary factor 1, beta Uterus 3.28 99032_at RASD1 RAS, dexamethasone-induced 1 Uterus 3.27 100428_at LAMC2 laminin, gamma 2 Uterus 3.25 103708_at UNK_AI132207 Cluster Incl AI132207: ue28g02.x1 Mus musculus cDNA, 3′ end/ Uterus 3.23 clone = IMAGE-1481714/clone_end = 3′/gb = AI132207/ gi = 3602223/ug = Mm.24090/len = 450/STRA = for 96693_at UNK_AI849453 ESTs, Highly similar to ARGINYL-TRNA SYNTHETASE Uterus 3.23 [Cricetulus longicaudatus] 94831_at CTSB cathepsin B Uterus 3.2 95493_at COL6A1 procollagen, type VI, alpha 1 Uterus 3.2 99562_at MAN2B1 mannosidase 2, alpha B1 Uterus 3.2 101487_f_at LY6E lymphocyte antigen 6 complex, locus E Uterus 3.18 100081_at STIP1 stress-induced phosphoprotein 1 Uterus 3.18 94061_at CRIP cysteine rich intestinal protein Uterus 3.18 101060_at GRP58 glucose regulated protein, 58 kDa Uterus 3.16 98522_at PSMD8 proteasome (prosome, macropain) 26S subunit, non-ATPase, 8 Uterus 3.16 101834_at MAPK3 mitogen activated protein kinase 3 Uterus 3.14 96657_at SAT spermidine/spermine N1-acetyl transferase Uterus 3.14 92632_at UNK_AI842328 Mus musculus calmodulin III (Calm3) mRNA, 3′ untranslated Uterus 3.12 region 99992_at UNK_AI286698 ESTs, Highly similar to interleukin 17 receptor□ [M. musculus] Uterus 3.12 94282_at ASAH1 N-acylsphingosine amidohydrolase 1 Uterus 3.11 94788_f_at TUBB5 tubulin, beta 5 Uterus 3.11 103398_at UNK_AW123232 Cluster Incl AW123232: UI-M-BH2.1-apd-g-08-0-UI.s1 Mus Uterus 3.1 musculus cDNA, 3′ end/clone = UI-M-BH2.1-apd-g-08-0-UI/ clone_end = 3′/gb = AW123232/gi = 6098727/ug = Mm.18714/ len = 469/STRA = rev 95149_at COPZ1 coatomer protein complex, subunit zeta 1 Uterus 3.1 103891_i_at UNK_AI197161 ESTs, Moderately similar to ELL2_HUMAN RNA Uterus 3.09 POLYMERASE II ELONGATION FACTOR ELL2□ [H. sapiens] 98104_at UNK_AI842889 ESTs, Highly similar to PROTEOLIPID PROTEIN PPA1 Uterus 3.08 [Saccharomyces cerevisiae] 102916_s_at CREBL1 cAMP responsive element binding protein-like 1 Uterus 3.08 101591_at UNK_AI852589 ESTs, Highly similar to HYPOTHETICAL PROTEIN Uterus 3.07 C22G7.01C IN CHROMOSOME I [Schizosaccharomyces pombe] 100949_at UNK_AI461767 ESTs, Weakly similar to hypothetical protein [H. sapiens] Uterus 3.05 96573_at ACTG actin, gamma, cytoplasmic Uterus 3.04 100948_at D15ERTD221E DNA segment, Chr 15, ERATO Doi 221, expressed Uterus 3.03 101754_f_at SPRR2G small proline-rich protein 2G Uterus 3.02 97829_at UNK_AI838053 ESTs, Highly similar to phosphatidylinositol synthase Uterus 3.02 [R. norvegicus] 101886_f_at H2-L histocompatibility 2, L region Uterus 3.01 99067_at GAS6 growth arrest specific 6 Uterus 3 101571_g_at IGFBP4 insulin-like growth factor binding protein 4 Uterus 3 100998_at H2-AB1 histocompatibility 2, class II antigen A, beta 1 Uterus 3 96135_at UNK_AA833425 ESTs, Highly similar to AF161398_1 HSPC280 [H. sapiens] Uterus 2.99 101105_at BCRP1-PENDING breakpoint cluster region protein 1 Uterus 2.97 96024_at AHCY S-adenosylhomocysteine hydrolase Uterus 2.96 100496_at PAM peptidylglycine alpha-amidating monooxygenase Uterus 2.95 103755_at SH3D19 SH3 domain protein D19 Uterus 2.95 97817_at SPEC1-PENDING small protein effector 1 of Cdc42 Uterus 2.95 98543_at CTSS cathepsin S Uterus 2.95 93548_at UNK_AW122942 ESTs, Highly similar to PROTEIN TRANSPORT PROTEIN Uterus 2.94 SEC61 BETA SUBUNIT [Homo sapiens; Canis familiaris] 97197_r_at UNK_C78850 Mouse (AKR/J) endogenous retrovirus, clone A-12, pol-env Uterus 2.93 region 102370_at UNK_AA822174 Cluster Incl AA822174: vp36a09.r1 Mus musculus cDNA, 5′ end/ Uterus 2.91 clone = IMAGE-1078744/clone_end = 5′/gb = AA822174/ gi = 2892042/ug = Mm.1187/len = 329/STRA = for 97559_at EEF2 eukaryotic translation elongation factor 2 Uterus 2.9 96732_at UNK_AI851081 ESTs, Highly similar to T17338 hypothetical protein Uterus 2.89 DKFZp434O125.1 - human [H. sapiens] 92450_at SLC12A4 solute carrier family 12, member 4 Uterus 2.88 93126_at CKB creatine kinase, brain Uterus 2.87 98417_at MX1 myxovirus (influenza virus) resistance 1 Uterus 2.87 96360_at UNK_AW125498 ESTs, Weakly similar to GDIS_MOUSE RHO GDP- Uterus 2.86 DISSOCIATION INHIBITOR 2 [M. musculus] 96356_at AF007010 EST AF007010 Uterus 2.84 95593_at UNK_AW125446 Cluster Incl AW125446: UI-M-BH2.3-aqh-h-05-0-UI.s1 Mus Uterus 2.84 musculus cDNA, 3′ end/clone = UI-M-BH2.3-aqh-h-05-0-UI/ clone_end = 3′/gb = AW125446/gi = 6100976/ug = Mm.27902/ len = 540/STRA = for 98405_at SPI6 serine protease inhibitor 6 Uterus 2.84 102804_at CEACAM1 CEA-related cell adhesion molecule 1 Uterus 2.83 92836_at UNK_AA919594 Cluster Incl AA919594: vz22b07.r1 Mus musculus cDNA, 5′ end/ Uterus 2.83 clone = IMAGE-1316437/clone_end = 5′/gb = AA919594/ gi = 3066373/ug = Mm.13097/len = 222/STRA = for 100460_at TSBP TPR-containing, SH2-binding phosphoprotein Uterus 2.83 100094_at SUPT5H suppressor of Ty 5 homolog (S. cerevisiae) Uterus 2.82 100064_f_at GJA1 gap junction membrane channel protein alpha 1 Uterus 2.81 96632_at MRGX-PENDING MORF-related gene X Uterus 2.81 95721_at MAPKAPK2 MAP kinase-activated protein kinase 2 Uterus 2.8 101948_at LAMB1-1 laminin B1 subunit 1 Uterus 2.8 101959_r_at TFDP1 transcription factor Dp 1 Uterus 2.79 97203_at MLP MARCKS-like protein Uterus 2.77 97496_f_at UNK_AW048944 ESTs, Weakly similar to polymerase I-transcript release factor Uterus 2.76 [M. musculus] 101877_at SLC31A1 solute carrier family 31, member 1 Uterus 2.76 104221_at SLC7A5 solute carrier family 7 (cationic amino acid transporter, y⁺ Uterus 2.75 system), member 5 98019_at TGFB1I1 transforming growth factor beta 1 induced transcript 1 Uterus 2.75 101510_at PSME1 protease (prosome, macropain) 28 subunit, alpha Uterus 2.74 96340_at UNK_AW124185 ESTs, Highly similar to C214_HUMAN 17.9 KDA MEMBRANE Uterus 2.74 PROTEIN C21ORF4□ [H. sapiens] 96761_at UNK_AF109906 Cluster Incl AF109906: Mus musculus MHC class III region RD Uterus 2.72 gene, partial cds; Bf, C2, G9A, NG22, G9, HSP70, HSP70, HSC70t, and smRNP genes, complete cds; G7A gene, partial cds; and unknown genes/cds = (0,2123)/gb = AF109906/ gi = 3986763/ug = Mm.29004/len = 212 102990_at COL3A1 procollagen, type III, alpha 1 Uterus 2.72 94224_s_at UNK_M74123 Mus musculus (strain C57BI/6) mRNA sequence Uterus 2.71 100621_at UNK_AI848825 ESTs, Highly similar to RIBONUCLEASE INHIBITOR [Rattus Uterus 2.7 norvegicus] 102752_at SHYC selective hybridizing clone Uterus 2.7 97013_f_at CYBA cytochrome b-245, alpha polypeptide Uterus 2.68 104248_at UNK_AW227650 ESTs, Highly similar to TRANSLOCON-ASSOCIATED Uterus 2.68 PROTEIN, GAMMA SUBUNIT [Rattus norvegicus] 104701_at STRA14 stimulated by retinoic acid 14 Uterus 2.68 103648_at TACSTD2 tumor-associated calcium signal transducer 2 Uterus 2.67 99514_at UNK_AI835443 ESTs, Highly similar to B-MYC TRANSFORMING PROTEIN Uterus 2.67 [Rattus norvegicus] 97262_at UNK_AW050305 ESTs, Highly similar to CASEIN KINASE I, DELTA ISOFORM Uterus 2.66 [Homo sapiens] 95694_at UNK_X70956 M. musculus TOP gene for topoisomerase I, exons 19-21 Uterus 2.66 101078_at BSG basigin Uterus 2.64 95660_at UNK_AI851815 Mus musculus HSCO mRNA, complete cds Uterus 2.63 99993_at ANPEP alanyl (membrane) aminopeptidase (aminopeptidase N, Uterus 2.63 aminopeptidase M, microsomal aminopeptidase, CD13, p150) 103494_at UNK_AI047972 ESTs, Weakly similar to CD63_MOUSE CD63 ANTIGEN□ Uterus 2.63 [M. musculus] 94929_at PTPN1 protein tyrosine phosphatase, non-receptor type 1 Uterus 2.6 100610_at CAPN4 calpain 4 Uterus 2.6 97890_at SGK serum/glucocorticoid regulated kinase Uterus 2.6 100889_at UNK_AI838576 Cluster Incl AI838576: UI-M-AO0-abz-c-02-0-UI.s1 Mus Uterus 2.59 musculus cDNA, 3′ end/clone = UI-M-AO0-abz-c-02-0-UI/ clone_end = 3′/gb = AI838576/gi = 5472789/ug = Mm.54120/ len = 181/STRA = rev 100475_at ZFP147 zinc finger protein 147 Uterus 2.59 98946_at WSB1 WSB-1 Uterus 2.59 96912_s_at CTLA2A cytotoxic T lymphocyte-associated protein 2 alpha Uterus 2.58 96069_at UNK_AI840094 ESTs, Highly similar to AFLATOXIN B1 ALDEHYDE Uterus 2.57 REDUCTASE [Rattus norvegicus] 100723_f_at SPRR2E small proline-rich protein 2E Uterus 2.57 93058_at EIF1A eukaryotic translation initiation factor 1A Uterus 2.56 94301_at ATP6K ATPase, H+ transporting lysosomal (vacuolar proton pump), Uterus 2.55 9.2 kDa 93680_at STK10 serine/threonine kinase 10 Uterus 2.55 93499_at CAPPA1 capping protein alpha 1 Uterus 2.55 100422_i_at UNK_AJ237939 Cluster Incl AJ237939: Mus musculus partial STAT5B gene, Uterus 2.53 exons 6-9/cds = (0,618)/gb = AJ237939/gi = 5689871/ ug = Mm.4697/len = 619/STRA = for 96333_g_at UNK_AW259199 ESTs, Weakly similar to AF154120_1 sorting nexin 1 Uterus 2.53 [M. musculus] 103918_at SLC15A2 solute carrier family 15 (H+/peptide transporter), member 2 Uterus 2.53 101982_at VASP vasodilator-stimulated phosphoprotein Uterus 2.53 104155_f_at ATF3 activating transcription factor 3 Uterus 2.53 96633_s_at MRGX-PENDING MORF-related gene X Uterus 2.52 95397_at UNK_AI852661 Cluster Incl AI852661: UI-M-BH0-aji-a-10-0-UI.s1 Mus Uterus 2.5 musculus cDNA, 3′ end/clone = UI-M-BH0-aji-a-10-0-UI/ clone_end = 3′/gb = AI852661/gi = 5496567/ug = Mm.2388/ len = 297/STRA = for 92809_r_at FKBP4 FK506 binding protein 4 (59 kDa) Uterus 2.5 100136_at LAMP2 lysosomal membrane glycoprotein 2 Uterus 2.5 93250_r_at HMGB2 high mobility group box 2 Uterus 2.48 103551_at AI428202 EST AI4282022 Uterus 2.48 100686_at LLREP3 repeat family 3 gene Uterus 2.46 92256_at CTSB cathepsin B Uterus 2.46 92226_at UNK_AA866971 ESTs, Moderately similar to hypothetical protein [H. sapiens] Uterus 2.45 96056_at ARHC aplysia ras-related homolog 9 (RhoC) Uterus 2.45 96920_at PRSS11 insulin-like growth factor binding protein 5 protease Uterus 2.44 101019_at CTSC cathepsin C Uterus 2.44 100600_at CD24A CD24a antigen Uterus 2.44 94915_at PPIB peptidylprolyl isomerase B Uterus 2.44 93323_at PLP2 proteolipid protein 2 Uterus 2.43 97386_at UNK_AI853294 Cluster Incl AI853294: UI-M-BH0-aji-f-03-0-UI.s1 Mus musculus Uterus 2.43 cDNA, 3′ end/clone = UI-M-BH0-aji-f-03-0-UI/clone_end = 3′/ gb = AI853294/gi = 5497200/ug = Mm.29789/len = 413/STRA = for 96939_at TRRP2 transient receptor protein 2 Uterus 2.43 95683_g_at DDB1 damage specific DNA binding protein 1 (127 kDa) Uterus 2.42 104292_at EYA2 eyes absent 2 homolog (Drosphila) Uterus 2.42 104300_at IQGAP1 IQ motif containing GTPase activating protein 1 Uterus 2.42 95120_at UNK_AI837621 ESTs, Highly similar to tetraspan NET-6 [H. sapiens] Uterus 2.41 98059_s_at LMNA lamin A Uterus 2.4 93320_at CPT1A carnitine palmitoyltransferase 1, liver Uterus 2.39 94260_at UNK_AI850352 ESTs, Moderately similar to KIAA0731 protein [H. sapiens] Uterus 2.39 94238_at UNK_AW228316 ESTs, Highly similar to serine protease [H. sapiens] Uterus 2.39 94206_at UNK_AC002397 Cluster Incl AC002397: Mouse chromosome 6 BAC-284H12 (Research Genetics mouse BAC library) complete sequence/ cds = (108,488)/gb = AC002397/gi = 3287367/ug = Mm.22195/ len = 568/STRA = for 93336_at UNK_AW121539 ESTs, Weakly similar to ENDOSOMAL P24B PROTEIN Uterus 2.38 PRECURSOR [Saccharomyces cerevisiae] 94060_at UNK_AI852623 ESTs, Weakly similar to Edp1 protein [M. musculus] Uterus 2.38 94834_at CTSH cathepsin H Uterus 2.37 101029_f_at ACTC1 actin, alpha, cardiac Uterus 2.37 100928_at FBLN2 fibulin 2 Uterus 2.37 92769_at TSTAP91A tissue specific transplantation antigen P91A Uterus 2.36 96829_at D19WSU162E DNA segment, Chr 19, Wayne State University 162, expressed Uterus 2.36 93309_at FIN14 fibroblast growth factor inducible 14 Uterus 2.36 101054_at II la-associated invariant chain Uterus 2.35 94839_at NUCB nucleobindin Uterus 2.35 98437_at CASP3 caspase 3, apoptosis related cysteine protease Uterus 2.34 98465_f_at IFI204 interferon activated gene 204 Uterus 2.33 98463_at REGULATOR ESTs, Highly similar to HOMEOTIC GENE REGULATOR Uterus 2.33 [DROSOPHILA [Drosophila melanogaster] MELANOGASTER] 103399_at SCML1 sex comb on midleg-like 1 (Drosophila) Uterus 2.32 94327_at UNK_AW230209 ESTs, Moderately similar to unnamed protein product [H. sapiens] Uterus 2.31 96345_at D2UCLA1 DNA segment, Chr 2, University of California at Los Angeles 1 Uterus 2.31 97751_f_at UNK_AI835771 ESTs, Moderately similar to G3P_MOUSE Uterus 2.3 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE [M. musculus] 101047_at VIM vimentin Uterus 2.3 100772_g_at LY57 lymphocyte antigen 57 Uterus 2.3 95128_at NCOR2 nuclear receptor co-repressor 2 Uterus 2.29 93101_s_at NEDD4 neural precursor cell expressed, developmentally down- Uterus 2.29 regulated gene 4 99119_at CFL1 cofilin 1, non-muscle Uterus 2.29 103341_at CTPS cytidine 5′-triphosphate synthase Uterus 2.28 92603_at ATP6D ATPase, H+ transporting, lysosomal (vacuolar proton pump), Uterus 2.28 42 kDa 96708_at UNK_AW120643 ESTs, Highly similar to COP-COATED VESICLE MEMBRANE Uterus 2.28 PROTEIN P24 PRECURSOR [Cricetulus griseus] 99100_at STAT3 signal transducer and activator of transcription 3 Uterus 2.28 95105_at UNK_AI847697 ESTs, Weakly similar to AF077034_1 HSPC010 [H. sapiens] Uterus 2.27 95142_s_at CAPPB1 capping protein beta 1 Uterus 2.27 94522_at DCTN3 dynactin 3 Uterus 2.26 98472_at H2-T23 histocompatibility 2, T region locus 23 Uterus 2.24 96025_g_at AHCY S-adenosylhomocysteine hydrolase Uterus 2.24 97689_at F3 coagulation factor III Uterus 2.23 104533_at UNK_AA764261 ESTs, Weakly similar to myelin transcription factor 1-like Uterus 2.23 [M. musculus] 104669_at IRF7 interferon regulatory factor 7 Uterus 2.21 97885_at 1810009M01RIK RIKEN cDNA 1810009M01 gene Uterus 2.21 92616_at UBE1X ubiquitin-activating enzyme E1, Chr X Uterus 2.21 93046_at NUP50 nucleoprotein 50 Uterus 2.2 98608_at D6ERTD109E DNA segment, Chr 6, ERATO Doi 109, expressed Uterus 2.19 92909_at PGF placental growth factor Uterus 2.19 101009_at KRT2-8 keratin complex 2, basic, gene 8 Uterus 2.19 100154_at D17WSU91E DNA segment, Chr 17, Wayne State University 91, expressed Uterus 2.19 96658_at UNK_AI841906 Cluster Incl AI841906: UI-M-AO0-acd-e-10-0-UI.s1 Mus Uterus 2.18 musculus cDNA, 3′ end/clone = UI-M-AO0-acd-e-10-0-UI/ clone_end3′/gb = AI841906/gi = 5476119/ug = Mm.27344/ len = 417/STRA = for 94018_at UBL3 ubiquitin-like 3 Uterus 2.18 98129_at ESET ERG-associated protein Uterus 2.17 98498_at CASP7 caspase 7 Uterus 2.17 94247_at ETS2 E26 avian leukemia oncogene 2, 3′ domain Uterus 2.16 100084_at VIL2 villin 2 Uterus 2.15 93093_at MCL1 myeloid cell leukemia sequence 1 Uterus 2.15 95109_at UNK_AW121447 ESTs, Weakly similar to SIK similar protein [M. musculus] Uterus 2.15 101963_at CTSL cathepsin L Uterus 2.14 102821_s_at RASL2-9 RAS-like, family 2, locus 9 Uterus 2.13 97240_g_at D19ERTD721E DNA segment, Chr 19, ERATO Doi 721, expressed Uterus 2.13 94257_at ARHGDIB rho, GDP dissociation inhibitor (GDI) beta Uterus 2.12 101543_f_at TUBA6 tubulin alpha 6 Uterus 2.11 100720_at PABPC1 poly A binding protein, cytoplasmic 1 Uterus 2.11 100566_at IGFBP5 insulin-like growth factor binding protein 5 Uterus 2.1 95647_f_at UNK_AI465845 ESTs, Moderately similar to unnamed protein product Uterus 2.1 [H. sapiens] 94899_at RHOIP3-PENDING Rho interacting protein 3 Uterus 2.09 104716_at RBP1 retinal binding protein 1, cellular Uterus 2.08 96338_at UNK_AW125059 ESTs, Weakly similar to A53770 growth factor-responsive Uterus 2.08 protein, vascular smooth muscle - rat□ [R. norvegicus] 103350_at PSMD7 proteasome (prosome, macropain) 26S subunit, non-ATPase, 7 Uterus 2.08 94040_at ERH enhancer of rudimentary homolog (Drosophila) Uterus 2.08 104041_at UNK_AW122255 ESTs, Moderately similar to T00076 hypothetical protein Uterus 2.07 KIAA0462 - human [H. sapiens] 96834_at UNK_AI843586 ESTs, Highly similar to PRE-MRNA SPLICING FACTOR SF2, Uterus 2.07 P33 SUBUNIT [Homo sapiens] 103955_at UNK_AW050325 ESTs, Highly similar to LAMBDA-CRYSTALLIN [Oryctolagus Uterus 2.07 cuniculus] 93997_at IFRG15 interferon alpha responsive protein (15 kDa) Uterus 2.06 99985_at TXNRD1 thioredoxin reductase 1 Uterus 2.06 104125_at HA1R-PENDING Hoxa1 regulated gene Uterus 2.05 92816_r_at EIF4A1 eukaryotic translation initiation factor 4A1 Uterus 2.05 98993_at PPP2R5C protein phosphatase 2, regulatory subunit B (B56), gamma Uterus 2.04 isoform 98113_at PSMB1 proteasome (prosome, macropain) subunit, beta type 1 Uterus 2.04 99566_at TPI triosephosphate isomerase Uterus 2.04 101107_at CALU calumenin Uterus 2.04 99599_s_at UNK_AW210320 ESTs, Weakly similar to AF121217_1 pro-alpha-2(I) collagen Uterus 2.03 [R. norvegicus] 96724_r_at D17H6S56E-5 DNA segment, Chr 17, human D6S56E 5 Uterus 2.03 97994_at TCF7 transcription factor 7, T-cell specific Uterus 2.03 95102_at UNK_AW123754 ESTs, Moderately similar to APB3_RAT AMYLOID BETA A4 Uterus 2.02 PRECURSOR PROTEIN-BINDING FAMILY A MEMBER 3 [R. norvegicus] 94454_at PRTB proline rich protein expressed in brain Uterus 2.02 103059_at FXYD3 FXYD domain-containing ion transport regulator 3 Uterus 2.02 93037_i_at ANXA1 annexin A1 Uterus 2.01 104385_i_at UNK_AI843901 Cluster Incl AI843901: UI-M-AK1-aeu-g-04-0-UI.s1 Mus Uterus 2.01 musculus cDNA, 3′ end/clone = UI-M-AK1-aeu-g-04-0-UI/ clone_end = 3′/gb = AI843901/gi = 5478114/ug = Mm.227/ len = 300/STRA = for 93490_at UNK_AI841771 ESTs, Weakly similar to contains similarity to Saccharomyces Uterus 2 cerevisiae MAF1 protein [C. elegans] 95406_at UNK_AW125347 Cluster Incl AW125347: UI-M-BH2.1-apy-h-03-0-UI.s1 Mus Uterus 1.99 musculus cDNA, 3′ end/clone = UI-M-BH2.1-apy-h-03-0-UI/ clone_end = 3′/gb = AW125347/gi = 6100877/ug = Mm.24219/ len = 331/STRA = for REPRESSIONS 93594_r_at EMP3 epithelial membrane protein 3 Uterus 0.55 104235_at VAMP2 vesicle-associated membrane protein 2 Uterus 0.54 97317_at ENPP2 ectonucleotide pyrophosphatase/phosphodiesterase 2 Uterus 0.52 94813_at GAS1 growth arrest specific 1 Uterus 0.51 95133_at ASNS asparagine synthetase Uterus 0.48 103353_f_at CYP4B1 cytochrome P450, subfamily IV B, polypeptide 1 Uterus 0.46 99577_at KITL kit ligand Uterus 0.45 102395_at PMP22 peripheral myelin protein, 22 kDa Uterus 0.44 93013_at IDB2 inhibitor of DNA binding 2 Uterus 0.44 101152_at HTR5A 5-hydroxytryptamine (serotonin) receptor 5A Uterus 0.41 92589_at UNK_AI846545 ESTs, Highly similar to SERB_HUMAN L-3-PHOSPHOSERINE Uterus 0.4 PHOSPHATASE [H. sapiens] 104217_at UNK_AW045753 Cluster Incl AW045753: UI-M-BH1-akt-a-10-0-UI.s1 Mus Uterus 0.39 musculus cDNA, 3′ end/clone = UI-M-BH1-akt-a-10-0-UI/ clone_end = 3′/gb = AW045753/gi = 5906282/ug = Mm.27893/ len = 407/STRA = rev 93503_at SDF5 stromal cell derived factor 5 Uterus 0.38 96672_at UNK_AW123564 ESTs, Weakly similar to S36166 paired box transcription factor Uterus 0.38 Pax-6 - rat [R. norvegicus] 93543_f_at GSTM1 glutathione S-transferase, mu 1 Uterus 0.36 93836_at BNIP3 BCL2/adenovirus E1B 19 kDa-interacting protein 1, NIP3 Uterus 0.35 98575_at FASN fatty acid synthase Uterus 0.33 99671_at ADN adipsin Uterus 0.33 101990_at LDH2 lactate dehydrogenase 2, B chain Uterus 0.3 98588_at FAH fumarylacetoacetate hydrolase Uterus 0.3 92592_at GDC1 glycerol phosphate dehydrogenase 1, cytoplasmic adult Uterus 0.3 104313_at UNK_AI842432 ESTs, Moderately similar to PHOSPHOGLUCOMUTASE Uterus 0.3 [Rattus norvegicus] 102094_f_at GSTM1 glutathione S-transferase, mu 1 Uterus 0.3 92202_g_at UNK_AI553024 ESTs, Highly similar to 2118318A promyelocyte leukemia Zn Uterus 0.29 finger protein [M. musculus] 94056_at SCD1 stearoyl-Coenzyme A desaturase 1 Uterus 0.27 95731_at UNK_AI843106 ESTs, Highly similar to p53 regulated PA26-T2 nuclear protein Uterus 0.27 [H. sapiens] 97844_at RGS2 regulator of G-protein signaling 2 Uterus 0.26 94516_f_at PENK2 preproenkephalin 2 Uterus 0.19 95082_at IGFBP3 insulin-like growth factor binding protein 3 Uterus 0.19 94057_g_at SCD1 stearoyl-Coenzyme A desaturase 1 Uterus 0.19 101560_at EMB embigin Uterus 0.18 93996_at CYP2E1 cytochrome P450, 2e1, ethanol inducible Uterus 0.18 101991_at FMO1 flavin containing monooxygenase 1 Uterus 0.17 92877_at TGFBI transforming growth factor, beta induced, 68 kDa Uterus 0.16 97402_at TEMT thioether S-methyltransferase Uterus 0.15 100567_at FABP4 fatty acid binding protein 4, adipocyte Uterus 0.14 99104_at ACRP30 adipocyte complement related protein of 30 kDa Uterus 0.13

TABLE IV Genes Regulated By Estrogen in the Kidney Study 1 Study 1 Approx WT ERbKO Ave Veh E2 Veh Expr Expr (/evansm/ (/evansm/ Kidney, Expr Kidney, mouse/Study (/evansm/Kidney, mouse/Study 1, mouse/Study 1, Poten- Poten- U74v2/WT 1, U74v2/KO tial tial Vehicle U74v2/WT Vehicle ERa ERb kidney E2 kidney kidney regs regs Fragment Name Exemplar Seq: A Unigene Known Gene.Name Fold (81010)) (81036)) (81025)) INDUCTIONS x 100060_l_at M13500 kallikrein 8 15.78 5 39 2 x 100061_f_at M13500 kallikrein 8 83.25 6 341 3 x 95775_f_at V00829 kallikrein 1 61.00 7 370 8 x 104495_f_at Y00500 Mm.30375 kallikrein 5 52.59 5 115 2 x 94716_f_at M17962 Mm.200410 kallikrein 9 44.67 16 380 8 x 100422_(—f)_at AJ237939 Mm.4697 signal transducer 39.21 5 151 2 and activator of transcription 5A x 100423_(—f)_at AJ237939 Mm.4697 signal transducer 3.80 41 96 13 and activator of transcription 5A x 101289_(—f)_at M17979 28.73 18 305 11 x 100681_(—f)_at V00829 25.24 8 123 6 x 94773_at X01801 nerve growth 17.80 5 53 2 factor, alpha x 104497_(—f)_at X03994 Mm.5193 kallikrein 8 17.44 6 53 3 x 168876_(—f)_at AV044014 Mm.143833 kallikrein 21 17.40 6 75 4 x 102693_(—f)_at J00389 Mm.143842 kallikrein 13, 17.24 5 86 6 kallikrein 26 x 100719_(—f)_at J03677 Mm.19214 kallikrein 16 14.45 13 119 7 x 114903_at AI447633 Mm.26357 13.56 10 96 7 x 139531_at AW120795 Mm.45188 12.37 2 40 2 x 101870_at V00793 7.39 5 21 2 x 104221_at AB017189 Mm.27943 solute carrier family 6.99 8 61 9 7 (cationic amino acid transporter, y+ system), member 5 x 95905_at AI118078 Mm.24361 6.67 15 83 7 x 111046_r_at AI957367 Mm.26838 17b dehydrogenase 6.34 2 17 2 A homolog x 133672_at AI451032 Mm.32389 5.91 2 11 2 x 117208_at AI838208 Mm.41330 RIKEN cDNA 5.86 3 37 3 1110003O08 gene x 96591_at U24703 Mm.3057 reelin 5.28 5 43 3 x 103048_at M12731 Mm.16469 neuroblastoma myc- 4.76 6 39 2 related oncogene 1 x 165248_f_at AV035328 Mm.37203 placental 4.50 6 38 6 lactogen 2 x 92232_at U88328 Mm.3468 cytokine inducible 4.37 5 45 6 SH-2 containing protein 3 x 164520_f_at AV302474 Mm.25743 Tmprss2 4.26 1 6 1 Transmembrane protease, serine 2 x 99632_at U83902 Mm.43444 MAD2 (mitotic 3.99 9 27 4 arrest deficient, homolog)-like 1 (yeast) x 116273_at AW123862 Mm.31953 3.91 5 7 5 x 131226_at AI842542 Mm.23157 3.60 2 8 2 x 104550_at AW123273 Mm.23710 ?CYP2S1 3.54 7 22 5 Cytochrome P450 x 105737_at AI851277 Mm.39735 3.38 2 8 1 x 100407_at L38580 Mm.4655 galanin 3.38 24 98 13 x 97353_at AI837497 Mm.29629 AF9Q34 NGAP- 3.35 13 29 4 like protein x 163941_at AI646761 Mm.202077 RIKEN cDNA 3.32 31 71 15 1110018J23 gene x 162976_at AI838662 Mm.198767 RIKEN cDNA 3.30 3 10 2 2700007F12 gene x 101979_at AF055638 Mm.9653 growth arrest and 3.26 8 42 8 DNA-damage- inducible 45 gamma x 93374_at AI836349 Mm.143762 junctophilin 3 3.23 25 65 6 x 107629_at AW048768 Mm.27667 3.17 14 34 13 x 112320_at AI852394 Mm.37753 3.15 14 30 8 x 133278_at AI452199 Mm.31771 3.11 3 8 2 x 95109_at AW121447 Mm.29363 NOL5A Nucleolar 3.07 33 91 21 protein 5A x 133815_at AU042854 Mm.26783 3.01 3 9 2 x 114394_at AW121080 Mm.32795 2.96 4 17 7 x 167969_at AA982630 Mm.87051 Weakly similar to 2.92 5 16 2 high mobility group 1 protein x 100938_at M31658 Mm.144157 growth hormone 2.88 9 41 7 releasing hormone x 97825_at AI854029 Mm.28209 p53 apoptosis 2.85 10 33 3 effector related to Pmp22 x 136270_at AI854101 Mm.40241 Highly similar 2.84 3 12 2 to CRFB MOUSE CORTICOTROPIN- RELEASING FACTOR BINDING PROTEIN PRECURSOR x 134303_at AI553493 Mm.35319 2.81 3 11 3 x 107993_at AI847249 Mm.27680 2.81 2 9 2 x 104776_at AA600617 Mm.33238 2.70 1 4 1 x 171390_i_at AV299689 Mm.21070 2.66 2 3 2 x 103556_at AI840158 Mm.19081 angiopoletin- 2.60 8 24 7 like 2 x 109176_at AI846059 Mm.29219 RAI17 Retinoic 2.58 47 79 33 acid Induced 17 x 163186_at AI852882 Mm.23230 RIKEN cDNA 2.57 20 61 25 2610510B01 gene x 170686_f_at AV362687 Mm.89845 2.52 22 58 27 x 164051_at AV359458 Mm.17850 2.46 87 195 90 x 108749_at AA611885 Mm.23047 2.41 16 34 12 x 96629_at X04097 Mm.27194 similar to 2.32 22 55 11 TESTOSTERONE- REGULATED RP2 PROTEIN x 110767_r_at AA959550 Mm.31540 vascular 2.31 13 37 9 endothetal growth factor x 165772_at AI851899 Mm.41409 RIKEN cDNA 2.24 17 29 13 0610039J01 gene x 99552_at U79550 Mm.4272 slug, chicken 2.23 7 19 6 homolog x 98437_at U83720 Mm.34405 caspase 3, apoptosis 2.22 21 39 11 related cysteine protease x 165710_at AA815844 sodium channel, 2.22 3 9 2 nonvoltage-gated 1 gamma x 110591_at AA270831 Mm.2454 SH3 domain 2.21 11 23 10 protien D19 x 113125_at AI851671 Mm.34064 signal transducer 2.20 11 31 11 and activator of transcription 5B x 110850_at AA959574 Mm.74711 NRIP1 Nuclear 2.18 28 76 25 receptor interacting protein 1, RIP140 x 103739_at AW230977 Mm.24411 RIKEN cDNA 2.16 10 33 10 1110017N23 gene x 165032_i_at AV365688 Mm.200980 RIKEN cDNA 2.15 20 42 12 4933429H19 gene x 166843_at AI851523 Mm.200318 2.12 149 174 105 x 166021_at AI481830 Mm.49448 2.09 7 12 5 x 100440_f_at U76758 Mm.4789 ankyrin 1, 2.08 60 119 19 erythroid x 163370_at AI591488 Mm.31024 Osbpl3 Oxysterol 2.08 6 10 7 binding protien-like 3 x 96215_f_at AI53421 Mm.218360 2.08 92 206 32 x 165866_f_at AV291803 Mm.70127 ribosomal 2.08 22 40 17 protein L12 x 98628_f_at AF003695 Mm.3879 hypoxia inducible 1.97 129 288 68 factor 1, alpha subunit REPRESSIONS x 104880_at AI843154 Mm.33750 0.50 10 2 11 x 129147_r_at AI931796 Mm.214530 similar to 0.50 20 6 15 TYROSINE- PROTEIN KINASE RECEPTOR TIE-1 PRECURSOR x 102788_s_at U70132 Mm.1385 paired-like 0.49 83 23 35 homeodomain transcription factor 2 x 166849_at AI853080 Mm.40718 0.49 40 16 29 x 165678_i_at AI482191 Mm.33178 0.49 23 11 15 x 106195_at AI851948 Mm.22808 0.48 17 8 15 x 131149_at AW214502 Mm.27650 RIKEN cDNA 0.48 17 7 16 5033417D07 gene x 162645_at AI851427 Mm.25594 protein kinase, 0.48 20 11 15 cAMP dependent regulatory, type II beta x 100539_at AI841279 Mm.157073 ?HBACH 0.47 38 16 22 Cytosolic acyl coenzyme A thioester hydrolase x 169068_i_at AV206066 Mm.59239 RIKEN cDNA 0.46 6 2 8 4930434J08 gene x 99127_at X61506 Mm.4098 spinocerebellar 0.45 136 66 52 ataxia 10 homolog (human) x 108265_at AW120464 Mm.54158 0.45 32 12 16 x 137525_at AI098139 Mm.38027 0.44 9 4 13 x 167023_f_at AV016619 Mm.2608 biglycan 0.44 15 3 6 x 101738_at U25145 Mm.57061 tutetnizing 0.43 1120 578 606 hormone beta x 104477_at AW047643 Mm.29940 0.43 23 15 18 x 93104_at Z16410 B-cell translocation 0.42 27 14 18 gene 1, anti- proliferative x 162969_at AW123298 Mm.41716 Edil3 EGF-like 0.41 55 34 41 repeats and discordin I-like domains 3 x 165569_at AI847273 Mm.22305 0.41 14 5 16 x 170896_at AV066592 Mm.34232 Immune 0.40 9 3 14 associated nucleotide 4 x 103729_at M36775 Mm.243 laminin, alpha 1 0.40 24 17 26 x 132403_at AI788603 Mm.169241 similar to 0.40 54 19 47 TSC1_RAT HAMARTIN (TUBEROUS SCLEROSIS 1 PROTEIN HOMOLOG) x 97519_at X13986 Mm.321 secreted 0.39 338 178 212 phosphoprotein 1 x 98055_at AW121500 Mm.34330 bladder cancer 0.39 33 14 19 associated protein homolog (human) x 163224_at AI843147 Mm.24577 IGSF1 0.38 51 27 33 Immunoglobulin superfamily, member 1 x 95559_at AI838836 Mm.27768 RIKEN cDNA 0.38 145 74 73 6330403K07 gene x 99057_at M12379 thymus cell 0.35 131 57 61 antigen 1, theta x 133139_at AW122295 Mm.41642 regulator of 0.34 20 4 22 G-protein signaling 4 x 162964_at AI854153 Mm.41842 regulator of 0.33 50 11 39 G-protein signaling 4 x 97520_s_at X83569 Mm.140956 neuroatin 0.32 523 157 314 x 94694_at M69196 Mm.1333 proprotein 0.29 99 27 51 convertase subtillsin/kexin type 1 x 108851_at AW125899 Mm.66275 Ras-like protein 0.27 33 10 33 x 101737_at U12932 Mm.46711 follicle stimulating 0.16 163 19 87 hormone beta Study 1 Study 2 Study 2 WT ERbKO E2 Veh Veh E2 E2 Veh Veh E2 E2 Expr Expr Expr Expr (/evansm/ (/evansm/ Expr Expr Expr (/evansm/ (/evansm/ Kidney, Kidney, (/evansm/ (/evansm/ (/evansm/Kidney, Kidney, Kidney, Expr mouse/Study mouse/Study Kidney, Kidney, mouse/Study mouse/Study mouse/Study (/evansm/Kidney, 2, 2, mouse/Study mouse/Study 2, 2, 2, mouse/Study U74v2/ER U74v2/ER 2, Poten- 1, U74v2/WT U74v2/WT U74v2/WT 2, bKO bKO U74v2/ER tial U74v2/KO kidney kidney kidney U74v2/WT kidney kidney bKO ERa E2 kidney vehicle vehicle E2 kidney E2 vehicle vehicle kidney E2 regs (81038)) (82406)) (82407)) (82408)) (82409)) (82410)) (82411)) (82412)) INDUCTIONS x 37 1 2 40 31 2 3 44 37 x 370 5 4 338 201 3 4 359 297 x 333 9 6 345 234 2 4 361 291 x 125 1 2 145 89 2 3 163 130 x 372 10 10 329 230 3 5 369 302 x 70 1 2 104 73 2 3 104 92 x 61 20 14 80 44 15 14 58 72 x 282 15 13 264 178 4 5 295 236 x 122 5 6 142 89 3 3 154 117 x 59 1 2 30 24 2 3 33 42 x 67 2 2 45 26 2 3 56 49 x 96 11 7 76 68 4 2 83 70 x 79 5 4 70 38 2 3 86 66 x 103 6 7 87 50 3 5 95 82 x 94 11 7 92 97 6 4 125 84 x 24 2 2 32 17 4 2 40 22 x 14 1 2 19 13 2 3 24 22 x 47 14 8 64 57 4 9 61 61 x 38 6 4 35 30 4 5 46 33 x 13 3 3 11 9 3 2 12 11 x 16 2 2 15 8 4 2 23 14 x 18 9 4 16 15 9 4 15 26 x 10 3 3 14 7 2 3 14 12 x 9 2 2 9 5 2 3 15 10 x 29 4 7 14 32 8 7 27 17 x 11 6 5 12 13 7 5 21 15 x 8 3 2 7 8 3 3 10 8 x 16 5 5 19 14 3 5 23 19 x 30 5 4 22 18 5 2 16 13 x 11 2 3 11 8 4 2 8 6 x 11 2 3 14 7 3 4 18 10 x 4 2 3 6 3 2 1 8 6 x 42 14 13 41 25 10 11 44 37 x 14 5 6 18 12 4 5 27 16 x 60 18 17 55 41 10 12 65 43 x 8 3 4 9 10 3 2 9 11 x 10 6 4 17 10 4 5 19 18 x 30 7 10 24 19 8 8 26 19 x 38 8 13 31 36 6 9 29 36 x 30 7 7 16 13 4 6 26 22 x 10 3 3 9 11 6 3 4 10 x 69 28 21 74 42 15 20 76 57 x 11 2 2 5 4 5 3 7 8 x 13 5 5 13 9 3 3 11 12 x 7 3 4 8 8 8 4 18 17 x 19 8 8 14 13 4 6 11 15 x 12 6 4 9 4 3 5 11 10 x 7 2 2 7 1 4 2 7 4 x 14 5 6 9 10 5 7 10 9 x 7 3 3 4 4 3 2 5 2 x 4 2 2 6 2 3 1 4 5 x 11 2 2 9 6 4 2 4 3 x 14 3 2 7 5 3 4 10 11 x 104 33 29 75 52 15 22 70 61 x 50 23 19 42 30 13 16 50 53 x 66 11 15 34 23 10 14 38 32 x 201 61 62 132 117 36 52 152 140 x 25 7 8 16 13 5 7 23 21 x 24 20 19 31 23 6 12 33 26 x 17 4 4 11 4 4 4 11 9 x 26 11 11 21 12 6 6 25 18 x 15 8 7 11 6 4 5 13 12 x 22 10 9 22 18 6 11 27 23 x 6 3 3 5 3 4 3 6 5 x 24 8 7 14 11 7 7 22 16 x 13 10 8 11 9 6 3 13 19 x 47 22 19 32 23 13 18 47 43 x 14 7 6 11 6 3 7 16 13 x 30 12 5 17 7 9 6 24 15 x 265 83 80 175 174 85 49 181 174 x 14 7 4 11 7 5 4 11 10 x 53 20 25 41 39 30 23 47 47 x 13 5 4 10 8 4 4 14 9 x 101 89 80 117 87 46 50 101 66 x 35 20 17 33 25 7 12 31 24 x 136 88 76 143 100 56 68 153 114 REPRESSIONS x 9 26 13 9 6 18 5 9 5 x 7 12 7 5 4 6 6 5 4 x 28 41 28 15 7 18 27 15 10 x 17 22 15 8 4 11 13 9 7 x 8 15 14 6 4 10 12 7 6 x 7 12 9 5 4 7 8 5 4 x 7 14 13 6 6 10 11 6 7 x 8 14 12 4 2 10 11 6 6 x 13 22 19 5 3 7 19 8 10 x 4 3 3 2 1 4 6 2 2 x 28 53 43 16 7 29 60 21 19 x 8 24 27 11 7 17 20 12 7 x 6 9 10 3 3 7 8 3 4 x 5 5 5 2 1 4 6 3 3 x 262 525 470 180 125 257 446 176 185 x 7 19 14 4 4 10 15 6 6 x 7 22 18 7 4 13 21 8 7 x 22 48 42 8 4 25 36 11 11 x 6 15 15 6 4 7 13 5 5 x 5 10 6 4 2 7 9 4 4 x 7 25 11 6 3 14 18 8 4 x 19 35 27 14 12 23 23 12 8 x 84 255 198 76 46 184 198 77 59 x 9 25 15 5 4 9 19 5 6 x 17 31 26 6 5 18 26 4 7 x 27 86 71 23 12 42 64 25 19 x 25 100 83 18 17 38 71 21 19 x 6 12 8 4 2 6 8 4 4 x 19 35 41 12 8 22 28 9 8 x 133 381 280 87 37 210 322 102 101 x 19 62 52 11 9 24 47 12 12 x 6 24 26 8 6 38 14 8 10 x 17 107 34 17 3 34 58 10 5

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1. A plurality of genes, each of whom is differentially expressed in kidney cells exposed to estrogen and/or other hormones or combination of hormones and kidney cells without said exposure, which plurality comprises a first group and a second group, wherein each gene in said first group is differentially expressed at a higher level in said kidney cells exposed to estrogen and/or other hormones or combination of hormones than in said kidney cells without said exposure, and wherein each gene in said second group is differentially expressed at a lower level in said kidney cells exposed to estrogen and/or other hormones or combination of hormones than in said kidney cells without said exposure.
 2. The plurality of claim 1, wherein said exposure is in vivo or in vitro.
 3. The plurality of claim 2, wherein said higher level and said lower level are assessed using a predetermined statistical significance standard based on measurements of expression levels.
 4. The plurality of claim 3, wherein said measurements are obtained using nucleotide arrays or nucleotide filters.
 5. The plurality of claim 4, wherein said first group comprises NTT73 and ABCC3.
 6. The plurality of claim 4, wherein said first group comprises CYP7B1.
 7. The plurality of claim 4, wherein said second group comprises BHMT and SAHH.
 8. The plurality of claim 4, wherein said first group comprises Tissue Factor, CYP7B1, BCAT1, STAT5A, and GADD45G, wherein said second group comprises BHMT.
 9. The plurality of claim 4, wherein said first group comprises CYP7B1, TF, SCYA28, Iga, Vk28, PHD 2, ELF 3, TIM1, STAT5A, COR1, BCAT1, ABCC3, TIM2, NAT6, RGS3, GNBP3, BCL7A, 17βDHH, FYVE ZFP, NTT73, AGPS, TRIM2, HBACH, CIS2, CYP27B1, and STAT5B, wherein said second group comprises SAHH, ADH1A7, RARRES2, and BHMT.
 10. A method for identifying an agent having the biological effect of estrogen and/or other hormones or combination of hormones on gene expression in kidney, wherein said desired effect represents a first plurality of genes differentially expressed at various levels, which method comprises: exposing, in vivo or in vitro, kidney cells to said agent; measuring expression levels of a multiplicity of genes in said kidney cells exposed to said agent and kidney cells without said exposure, said multiplicity being greater than said first plurality; determining, using a predetermined statistical significance standard, genes which are differentially expressed in said kidney cells exposed to said agent and said kidney cells without said exposure, said genes constitute a second plurality; and comparing the expression levels of genes in said second plurality with the expression levels of genes in said first plurality, wherein said agent is identified as having said desired effort if said first and second pluralities are the same and said expression levels in said first and second pluralities are substantially the same.
 11. The method of claim 10, wherein said measuring is performed using nucleotide arrays or nucleotide filters.
 12. The method of claim 11, wherein said comparing is performed using a suitable statistical technique.
 13. The method of claim 12, wherein said first plurality is the plurality of any of claims 5-9.
 14. The method of claim 11, wherein said first plurality is the plurality of any of claim 5-9.
 15. An agent identified by the method of claim
 13. 16. An agent identified by the method of claim
 14. 17. A pharmaceutical composition comprising the agent of claim 15 and a pharmaceutically acceptable excipient.
 18. A pharmaceutical composition comprising the agent of claim 16 and a pharmaceutically acceptable excipient.
 19. A method for identifying an agent capable of maintaining vascular volume in septic shock, which method comprises: exposing, in vivo or in vitro, kidney cells to said agent; measuring expression levels of NTT73 and ABCC3 in said kidney cells exposed to said agent and kidney cells without said exposure; comparing the expression levels of NTT73 and ABCC3 with the expression levels of genes in the plurality of claim 5, wherein said agent is identified as capable of maintaining vascular volume in septic shock if said expression levels of NTT73 and ABCC3 are substantially the same as said expression levels of genes in the plurality of claim
 5. 20. A method for identifying an agent capable of enhancing calcium uptake in post-menopausal women, which method comprises: exposing, in vivo or in vitro, kidney cells to said agent; measuring expression levels of CYP7B1 in said kidney cells exposed to said agent and kidney cells without said exposure; comparing the expression levels of CYP7B1 with the expression levels of genes in the plurality of claim 6, wherein said agent is identified as capable of enhancing calcium uptake in post-menopausal women if said expression levels of CYP7B1 are substantially the same as said expression levels of genes in the plurality of claim
 6. 21. A method for identifying an agent for treating cardiovascular disorders, which method comprises: exposing, in vivo or in vitro, kidney cells to said agent; measuring expression levels of BHMT and SAHH in said kidney cells exposed to said agent and kidney cells without said exposure; comparing the expression levels of BHMT and SAHH with the expression levels of genes in the plurality of claim 7, wherein said agent is identified for treating cardiovascular disorders if said expression levels of BHMT and SAHH are substantially the same as said expression levels of genes in the plurality of claim
 7. 22. The method of claim 19, 20 or 21, wherein said measuring is performed using nucleotide arrays or nucleotide filters.
 23. The method of claim 22, wherein said comparing is performed using a suitable statistical technique.
 24. An agent identified by the method of claim
 21. 25. An agent identified by the method of claim
 22. 26. A pharmaceutical composition comprising the agent of claim 24 and a pharmaceutically acceptable excipient.
 27. A pharmaceutical composition comprising the agent of claim 25 and a pharmaceutically acceptable excipient.
 28. A plurality of genes, each of whom is differentially expressed in pituitary cells exposed to estrogen and/or a hormone or combination of hormones and pituitary cells without said exposure, which plurality comprises a first group and a second group, wherein each gene in said first group is differentially expressed at a higher level in said pituitary cells exposed to estrogen and/or other hormones or combination of hormones than in said pituitary cells without said exposure, wherein each gene in said second group is differentially expressed at a lower level in said pituitary cells exposed to estrogen and/or other hormones or combination of hormones than in said pituitary cells without said exposure.
 29. The plurality of claim 28, wherein said exposure is in vivo or in vitro.
 30. The plurality of claim 29, wherein said higher level and said lower level are assessed using a predetermined statistical significance standard based on measurements of expression levels.
 31. The plurality of claim 30, wherein said measurements are obtained using nucleotide arrays or nucleotide filters.
 32. The plurality of claim 31, wherein said first group comprises STAT5B and GADD45G.
 33. The plurality of claim 31, wherein said first group comprises STAT5B, GADD45G1, and Kallikreins.
 34. The plurality of claim 31, 32 or 33, wherein said second group comprises FSHb.
 35. A method for identifying an agent having a desired effect of estrogen and/or other hormones or combination of hormones on gene expression in pituitary, wherein said desired effect represents a first plurality of genes differentially expressed at various levels, which method comprises: exposing, in vivo or in vitro, pituitary cells to said agent; measuring expression levels of a multiplicity of genes in said pituitary cells exposed to said agent and pituitary cells without said exposure, said multiplicity being greater than said first plurality; determining, using a predetermined statistical significance standard, genes which are differentially expressed in said pituitary cells exposed to said agent and said pituitary cells without said exposure, said genes constitute a second plurality; and comparing the expression levels of genes in said second plurality with the expression levels of genes in said first plurality, wherein said agent is identified as having said desired effort if said first and second pluralities are the same and said expression levels in said first and second pluralities are substantially the same.
 36. The method of claim 35, wherein said measuring is performed using nucleotide arrays or nucleotide filters.
 37. The method of claim 36, wherein said comparing is performed using a suitable statistical technique.
 38. An agent identified by the method of claim
 35. 39. A pharmaceutical composition comprising the agent of claim 38, and a pharmaceutically acceptable excipient.
 40. A plurality of genes, each of whom is differentially expressed in uterus cells exposed to estrogen and/or a hormone or combination of hormones and uterus cells without said exposure, which plurality comprises a first group and a second group, wherein each gene in said first group is differentially expressed at a higher level in said uterus cells exposed to estrogen and/or other hormones or combination of hormones than in said uterus cells without said exposure, wherein each gene in said second group is differentially expressed at a lower level in said uterus cells exposed to estrogen and/or other hormones or combination of hormones than in said uterus cells without said exposure.
 41. The plurality of claim 40, wherein said exposure is in vivo or in vitro.
 42. The plurality of claim 41, wherein said higher level and said lower level are assessed using a predetermined statistical significance standard based on measurements of expression levels.
 43. The plurality of claim 42, wherein said measurements are obtained using nucleotide arrays or nucleotide filters.
 44. The plurality of claim 43, wherein said first group comprises SFRP4, Deiodinase, type II, Procollagen, type I, alpha I, vimentin, and IDFBP4.
 45. The plurality of claim 43, wherein said first group comprises AI121305, ALOX15, BCAT1, SiAMOX, C3, FOS, MAP2k1, CEBPb, and EGR1.
 46. The plurality of claim 43, wherein said first group comprises SFRP4, Deiodinase (type II), Procollagen (ype I, alpha I) vimentin, IDFBP4, AI121305, ALOX15, BCAT1, SiAMOX, C3, FOS, MAP2k1, CEBPb, and EGR1.
 47. The plurality of claim 43, 44, 45 or 46, wherein said second group comprises CYP1A1.
 48. The plurality of claim 43, 44, 45, or 46, wherein said second group comprises Scavenger receptor.
 49. The plurality of claim 43, 44, 45, or 46, wherein said second group comprises CYP1A1 and Scavenger receptor.
 50. A method for identifying an agent having a desired effect of estrogen and/or other hormone or combination of hormones on gene expression in uterus, wherein said desired effect represents a first plurality of genes differentially expressed at various levels, which method comprises: exposing, in vivo or in vitro, uterus cells to said agent; measuring expression levels of a multiplicity of genes in said uterus cells exposed to said agent and uterus cells without said exposure, said multiplicity being greater than said first plurality; determining, using a predetermined statistical significance standard, genes which are differentially expressed in said uterus cells exposed to said agent and said uterus cells without said exposure, said genes constitute a second plurality; and comparing the expression levels of genes in said second plurality with the expression levels of genes in said first plurality, wherein said agent is identified as having said desired effort if said first and second pluralities are the same and said expression levels in said first and second pluralities are substantially the same.
 51. The method of claim 50, wherein said measuring is performed using nucleotide arrays or nucleotide filters.
 52. The method of claim 51, wherein said comparing is performed using a suitable statistical technique.
 53. An agent identified by the method of claim
 50. 54. A pharmaceutical composition comprising the agent of claim 53, and a pharmaceutically acceptable excipient.
 55. A plurality of genes of any one of claims 1, 28 or 40, wherein said expression levels are confirmed by real-time PCR.
 56. The method of identifying of any of claims 10, 19, 20, 21, 35 or 50 wherein said expression levels are confirmed by real-time PCR.
 57. A solid substrate comprising the plurality of genes of one of claims 1, 28 or
 40. 58. The solid substrate of claim 55, which is a gene chip.
 59. A kit comprising the plurality of genes of one of claims 1, 28 or
 40. 